The impact of the new 36 V lead-acid battery systems on lead consumption

NASA Astrophysics Data System (ADS)

Prengaman, R. David

The production of vehicles utilizing 36 V battery systems has begun with the introduction of the Toyota Crown. Other vehicles with 36 V batteries are in the near horizon. These vehicles may contain single or dual battery systems. These vehicles will most likely contain valve-regulated lead-acid (VRLA) batteries. The battery systems developed to date utilize significantly more lead than conventional 12 V batteries. This paper will evaluate the different proposed 36 V battery systems and estimate the lead requirements for each of the competing systems. It will also project the penetration of and resultant increased lead usage of these new batteries into the future.

Micro-hybrid electric vehicle application of valve-regulated lead-acid batteries in absorbent glass mat technology: Testing a partial-state-of-charge operation strategy

NASA Astrophysics Data System (ADS)

Schaeck, S.; Stoermer, A. O.; Hockgeiger, E.

The BMW Group has launched two micro-hybrid functions in high volume models in order to contribute to reduction of fuel consumption in modern passenger cars. Both the brake energy regeneration (BER) and the auto-start-stop function (ASSF) are based on the conventional 14 V vehicle electrical system and current series components with only little modifications. An intelligent control algorithm of the alternator enables recuperative charging in braking and coasting phases, known as BER. By switching off the internal combustion engine at a vehicle standstill the idling fuel consumption is effectively reduced by ASSF. By reason of economy and package a lead-acid battery is used as electrochemical energy storage device. The BMW Group assembles valve-regulated lead-acid (VRLA) batteries in absorbent glass mat (AGM) technology in the micro-hybrid electrical power system since special challenges arise for the batteries. By field data analysis a lower average state-of-charge (SOC) due to partial state-of-charge (PSOC) operation and a higher cycling rate due to BER and ASSF are confirmed in this article. Similar to a design of experiment (DOE) like method we present a long-term lab investigation. Two types of 90 Ah VRLA AGM batteries are operated with a test bench profile that simulates the micro-hybrid vehicle electrical system under varying conditions. The main attention of this lab testing is focused on capacity loss and charge acceptance over cycle life. These effects are put into context with periodically refresh charging the batteries in order to prevent accelerated battery aging due to hard sulfation. We demonstrate the positive effect of refresh chargings concerning preservation of battery charge acceptance. Furthermore, we observe moderate capacity loss over 90 full cycles both at 25 °C and at 3 °C battery temperature.

ABLE project: Development of an advanced lead-acid storage system for autonomous PV installations

NASA Astrophysics Data System (ADS)

Lemaire-Potteau, Elisabeth; Vallvé, Xavier; Pavlov, Detchko; Papazov, G.; Borg, Nico Van der; Sarrau, Jean-François

In the advanced battery for low-cost renewable energy (ABLE) project, the partners have developed an advanced storage system for small and medium-size PV systems. It is composed of an innovative valve-regulated lead-acid (VRLA) battery, optimised for reliability and manufacturing cost, and an integrated regulator, for optimal battery management and anti-fraudulent use. The ABLE battery performances are comparable to flooded tubular batteries, which are the reference in medium-size PV systems. The ABLE regulator has several innovative features regarding energy management and modular series/parallel association. The storage system has been validated by indoor, outdoor and field tests, and it is expected that this concept could be a major improvement for large-scale implementation of PV within the framework of national rural electrification schemes.

Development of a lead-acid battery for a hybrid electric vehicle

NASA Astrophysics Data System (ADS)

Cooper, A.

In September 2000, a project reliable, highly optimized lead-acid battery (RHOLAB) started under the UK Foresight Vehicle Programme with the objective of developing an optimized lead-acid battery solution for hybrid electric vehicles. The work is based on a novel, individual, spirally-wound valve-regulated lead-acid 2 V cell optimized for HEV use and low variability. This cell is being used as a building block for the development of a complete battery pack that is managed at the cell level. Following bench testing, this battery pack is to be thoroughly evaluated by substituting it for the Ni-MH pack in a Honda Insight. The RHOLAB cell is based on the 8 Ah Hawker Cyclon cell which has been modified to have current take-off at both ends—the dual-tab design. In addition, a variant has been produced with modified cell chemistry to help deal with problems that can occur when these valve-regulated lead-acid battery (VRLA) cells operate in a partial-state-of-charge condition. The cells have been cycled to a specially formulated test cycle based on real vehicle data derived from testing the Honda Insight on the various test tracks at the Millbrook Proving Grounds in the UK. These cycling tests have shown that the lead-acid pack can be successfully cycled when subjected to the high current demands from the vehicle, which have been measured at up to 15 C on discharge and 8 C during regenerative recharging, and cycle life is looking very promising under this arduous test regime. Concurrent with this work, battery development has been taking place. It was decided early on to develop the 144 V battery as four 36 V modules. Data collection and control has been built-in and special steps taken to minimize the problems of interconnect in this complex system. Development of the battery modules is now at an advanced stage. The project plan then allows for extensive testing of the vehicle with its lead-acid battery at Millbrook so it can be compared with the benchmark tests which have already been carried out on the vehicle with its Ni-MH batteries.

A field operational test on valve-regulated lead-acid absorbent-glass-mat batteries in micro-hybrid electric vehicles. Part I. Results based on kernel density estimation

NASA Astrophysics Data System (ADS)

Schaeck, S.; Karspeck, T.; Ott, C.; Weckler, M.; Stoermer, A. O.

2011-03-01

In March 2007 the BMW Group has launched the micro-hybrid functions brake energy regeneration (BER) and automatic start and stop function (ASSF). Valve-regulated lead-acid (VRLA) batteries in absorbent glass mat (AGM) technology are applied in vehicles with micro-hybrid power system (MHPS). In both part I and part II of this publication vehicles with MHPS and AGM batteries are subject to a field operational test (FOT). Test vehicles with conventional power system (CPS) and flooded batteries were used as a reference. In the FOT sample batteries were mounted several times and electrically tested in the laboratory intermediately. Vehicle- and battery-related diagnosis data were read out for each test run and were matched with laboratory data in a data base. The FOT data were analyzed by the use of two-dimensional, nonparametric kernel estimation for clear data presentation. The data show that capacity loss in the MHPS is comparable to the CPS. However, the influence of mileage performance, which cannot be separated, suggests that battery stress is enhanced in the MHPS although a battery refresh function is applied. Anyway, the FOT demonstrates the unsuitability of flooded batteries for the MHPS because of high early capacity loss due to acid stratification and because of vanishing cranking performance due to increasing internal resistance. Furthermore, the lack of dynamic charge acceptance for high energy regeneration efficiency is illustrated. Under the presented FOT conditions charge acceptance of lead-acid (LA) batteries decreases to less than one third for about half of the sample batteries compared to new battery condition. In part II of this publication FOT data are presented by multiple regression analysis (Schaeck et al., submitted for publication [1]).

VRLA Ultrabattery for high-rate partial-state-of-charge operation

NASA Astrophysics Data System (ADS)

Lam, L. T.; Louey, R.; Haigh, N. P.; Lim, O. V.; Vella, D. G.; Phyland, C. G.; Vu, L. H.; Furukawa, J.; Takada, T.; Monma, D.; Kano, T.

The objective of this study is to produce and test the hybrid valve-regulated Ultrabattery designed specifically for hybrid-electric vehicle duty, i.e., high-rate partial-state-of-charge operation. The Ultrabattery developed by CSIRO Energy Technology is a hybrid energy-storage device, which combines an asymmetric supercapacitor, and a lead-acid battery in one unit cells, taking the best from both technologies without the need for extra, expensive electronic controls. The capacitor will enhance the power and lifespan of the lead-acid battery as it acts as a buffer during high-rate discharging and charging. Consequently, this hybrid technology is able to provide and absorb charge rapidly during vehicle acceleration and braking. The work programme of this study is divided into two main parts, namely, field trial of prototype Ultrabatteries in a Honda Insight HEV and laboratory tests of prototype batteries. In this paper, the performance of prototype Ultrabatteries under different laboratory tests is reported. The evaluation of Ultrabatteries in terms of initial performance and cycling performance has been conducted at both CSIRO and Furukawa laboratories. The initial performance of prototype Ultrabatteries, such as capacity, power, cold cranking and self-discharge has been evaluated based upon the US FreedomCAR Battery Test Manual (DOE/ID-11069, October 2003). Results show that the Ultrabatteries meet, or exceed, respective targets of power, available energy, cold cranking and self-discharge set for both minimum and maximum power-assist HEVs. The cycling performance of prototype Ultrabatteries has been evaluated using: (i) simplified discharge and charge profile to simulate the driving conditions of micro-HEV; (ii) 42-V profile to simulate the driving conditions of mild-HEV and (iii) EUCAR and RHOLAB profiles to simulate the driving conditions of medium-HEV. For comparison purposes, nickel-metal-hydride (Ni-MH) cells, which are presently used in the Honda Insight HEV, have also been subjected to some of the above profiles (i.e., simplified discharge and charge profile and EUCAR profile). Although the Ultrabattery and a Ni-MH cell under EUCAR test profile are still on cycling, the outcomes to date show that the performance of these batteries and cells has been at least four times longer than that of the state-of-the art lead-acid cells or batteries. Excitingly, the performance of Ultrabatteries is proven to be comparable with that of the Ni-MH cells.

Impedance-based overcharging and gassing model for VRLA/AGM batteries

NASA Astrophysics Data System (ADS)

Thele, M.; Karden, E.; Surewaard, E.; Sauer, D. U.

This paper presents for the first time an impedance-based non-linear model for lead-acid batteries that is applicable in all operational modes. An overcharging model describes the accumulation and depletion of the dissolved Pb 2+ ions. This physical model has been added to the earlier presented model to expand the model validity. To properly represent the charge acceptance during dynamic operation, a concept of "hardening crystals" has been introduced in the model. Moreover, a detailed gassing and oxygen recombination model has been integrated. A realistic simulation of the overcharging behavior is now possible. The mathematical description is given in the paper. Simplifications are introduced that allow for an efficient implementation and for model parameterization in the time domain. A comparison between experimental data and simulation results demonstrates the achieved accuracy. The model enhancement is of major importance to analyze charging strategies especially in partial-cycling operation with limited charging time, e.g. in electrically assisted or hybrid cars and autonomous power supply systems.

Development of new positive-grid alloy and its application to long-life batteries for automotive industry

NASA Astrophysics Data System (ADS)

Furukawa, Jun; Nehyo, Y.; Shiga, S.

Positive-grid corrosion and its resulting creep or growth is one of the major causes of the failure of automotive lead-acid batteries. The importance of grid corrosion and growth is increasing given the tendency for rising temperatures in the engine compartments of modern vehicles. In order to cope with this situation, a new lead alloy has been developed for positive-grids by utilizing an optimized combination of lead-calcium-tin and barium. In addition to enhanced mechanical strength at high temperature, the corrosion-resistance of the grid is improved by as much as two-fold so that the high temperature durability of batteries using such grids has been demonstrated in both hot SAE J240 tests and in field trials in Japan and Thailand. A further advantage of the alloy is its recycleability compared with alloys containing silver. The new alloy gives superior performance in both 12-V flooded and 36-V valve-regulated lead-acid (VRLA) batteries.

Advanced valve-regulated lead-acid batteries for hybrid vehicle applications

NASA Astrophysics Data System (ADS)

Soria, M. L.; Trinidad, F.; Lacadena, J. M.; Sánchez, A.; Valenciano, J.

Future vehicle applications require the development of reliable and long life batteries operating under high-rate partial-state-of-charge (HRPSoC) working conditions. Work presented in this paper deals with the study of different design parameters, manufacturing process and charging conditions of spiral wound valve-regulated lead-acid (VRLA) batteries, in order to improve their reliability and cycle life for hybrid vehicle applications. Test results show that both electrolyte saturation and charge conditions have a strong effect on cycle life at HRPSoC performance, presumably because water loss finally accelerates battery failure, which is linked to irreversible sulphation in the upper part of the negative electrodes. By adding expanded graphite to the negative active mass formulation, increasing the electrolyte saturation degree (>95%) and controlling overcharge during regenerative braking periods (voltage limitation and occasional boosting) it is possible to achieve up to 220,000 cycles at 2.5% DOD, equivalent to 5500 capacity throughput. These results could make lead acid batteries a strong competitor for HEV applications versus other advanced systems such as Ni-MH or Li-ion batteries.

The Asian battery market—a decade of change

NASA Astrophysics Data System (ADS)

Eckfeld, S.; Manders, J. E.; Stevenson, M. W.

The Asian battery industry will undergo significant change over the next decade as it adapts to the enormous economic and technological pressures of our rapidly changing world. Europe and North America in recent years have seen significant rationalisation in battery manufacturing capacity and ownership for a variety of reasons. Into the future, Asia will be no exception, but the rate and magnitude of change may conceivably be greater than that already experienced elsewhere. Rationalisation in battery manufacturing plants will occur as a result of the establishment of super plants to manufacture batteries in order to improve the economies of scale and to facilitate the heavy investment in new capital and equipment that will be required to supply the newer technology battery types. The impact of 42 V automotive systems and valve-regulated lead-acid (VRLA) batteries will be influential on this scenario. It is expected that China, Japan, South Korea, and Thailand will feature heavily in the future Asian battery scene at the expense of some established countries and producers. The current state of the battery industry in Asia, factors driving change in Asia, and the likely implications for those companies that are currently manufacturing batteries in Asia or considering a future role in Asia within the coming decade are examined in this paper.

Deconstructing three-dimensional (3D) structure of absorptive glass mat (AGM) separator to tailor pore dimensions and amplify electrolyte uptake

NASA Astrophysics Data System (ADS)

Rawal, Amit; Rao, P. V. Kameswara; Kumar, Vijay

2018-04-01

Absorptive glass mat (AGM) separator is a vital technical component in valve regulated lead acid (VRLA) batteries that can be tailored for a desired application. To selectively design and tailor the AGM separator, the intricate three-dimensional (3D) structure needs to be unraveled. Herein, a toolkit of 3D analytical models of pore size distribution and electrolyte uptake expressed via wicking characteristics of AGM separators under unconfined and confined states is presented. 3D data of fiber orientation distributions obtained previously through X-ray micro-computed tomography (microCT) analysis are used as key set of input parameters. The predictive ability of pore size distribution model is assessed through the commonly used experimental set-up that usually apply high level of compressive stresses. Further, the existing analytical model of wicking characteristics of AGM separators has been extended to account for 3D characteristics, and subsequently, compared with the experimental results. A good agreement between the theory and experiments pave the way to simulate the realistic charge-discharge modes of the battery by applying cyclic loading condition. A threshold criterion describing the invariant behavior of pore size and wicking characteristics in terms of maximum permissible limit of key structural parameters during charge-discharge mode of the battery has also been proposed.

Further demonstration of the VRLA-type UltraBattery under medium-HEV duty and development of the flooded-type UltraBattery for micro-HEV applications

NASA Astrophysics Data System (ADS)

Furukawa, J.; Takada, T.; Monma, D.; Lam, L. T.

The UltraBattery has been invented by the CSIRO Energy Technology in Australia and has been developed and produced by the Furukawa Battery Co., Ltd., Japan. This battery is a hybrid energy storage device which combines a super capacitor and a lead-acid battery in single unit cells, taking the best from both technologies without the need of extra, expensive electronic controls. The capacitor enhances the power and lifespan of the lead-acid battery as it acts as a buffer during high-rate discharging and charging, thus enabling it to provide and absorb charge rapidly during vehicle acceleration and braking. The laboratory results of the prototype valve-regulated UltraBatteries show that the capacity, power, available energy, cold cranking and self-discharge of these batteries have met, or exceeded, all the respective performance targets set for both minimum and maximum power-assist HEVs. The cycling performance of the UltraBatteries under micro-, mild- and full-HEV duties is at least four times longer than that of the state-of-the-art lead-acid batteries. Importantly, the cycling performance of UltraBatteries is proven to be comparable or even better than that of the Ni-MH cells. On the other hand, the field trial of UltraBatteries in the Honda Insight HEV shows that the vehicle has surpassed 170,000 km and the batteries are still in a healthy condition. Furthermore, the UltraBatteries demonstrate very good acceptance of the charge from regenerative braking even at high state-of-charge, e.g., 70% during driving. Therefore, no equalization charge is required for the UltraBatteries during field trial. The HEV powered by UltraBatteries gives slightly higher fuel consumption (cf., 4.16 with 4.05 L/100 km) and CO 2 emissions (cf., 98.8 with 96 g km -1) compared with that by Ni-MH cells. There are no differences in driving experience between the Honda Insight powered by UltraBatteries and by Ni-MH cells. Given such comparable performance, the UltraBattery pack costs considerably less - only 20-40% of that of the Ni-MH pack by one estimate. In parallel with the field trial, a similar 144-V valve-regulated UltraBattery pack was also evaluated under simulated medium-HEV duty in our laboratories. In this study, the laboratory performance of the 144-V valve-regulated UltraBattery pack under simulated medium-HEV duty and that of the recently developed flooded-type UltraBattery under micro-HEV duty will be discussed. The flooded-type UltraBattery is expected to be favorable to the micro-HEVs because of reduced cost compared with the equivalent valve-regulated counterpart.

USABC Development of 12 Volt Battery for Start-Stop Application: Preprint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tataria, H.; Gross, O.; Bae, C.

Global automakers are accelerating the development of fuel efficient vehicles, as a part of meeting regional regulatory CO2 emissions requirements. The micro hybrid vehicles with auto start-stop functionality are considered economical solutions for the stringent European regulations. Flooded lead acid batteries were initially considered the most economical solution for idle-stop systems. However, the dynamic charge acceptance (DCA) at lower state-of-charge (SOC) was limiting the life of the batteries. While improved lead-acid batteries with AGM and VRLA features have improved battery longevity, they do not last the life of the vehicle. The United States Advanced Battery Consortium (or USABC, a consortiummore » of GM, Ford, and Chrysler) analyzed energy storage needs for a micro hybrid automobile with start-stop capability, and with a single power source. USABC has analyzed the start-stop behaviors of many drivers and has developed the requirements for the start-stop batteries (Table 3). The testing procedures to validate the performance and longevity were standardized and published. The guideline for the cost estimates calculations have also been provided, in order to determine the value of the newly developed modules. The analysis effort resulted in a set of requirements which will help the battery manufacturers to develop a module to meet the automotive Original Equipment Manufacturers (OEM) micro hybrid vehicle requirements. Battery developers were invited to submit development proposals and two proposals were selected for 50% cost share with USABC/DOE.« less

Development of high power VRLA batteries using novel materials and processes

NASA Astrophysics Data System (ADS)

Soria, M. L.; Valenciano, J.; Ojeda, A.; Raybaut, G.; Ihmels, K.; Deiters, J.; Clement, N.; Morales, J.; Sánchez, L.

Nowadays UPS manufacturers demand batteries with very high specific power and relatively low specific energy, because most mains failures can be defined as "microfailures", usually of the order of seconds. Due to this fact, it is not necessary to provide energy but power. Within a 3-year EU funded project, a new AGM valve-regulated lead-acid battery with weight and volume substantially reduced, as well as a substantial improvement in its reliability, is under development. These aspects can provide the achievement of a more efficient, safe and economic energy supply. Battery specific power is practically related to electrode area, so that its increase, and therefore an electrode thickness reduction, appear essential to achieve the project objectives. Furthermore, it is necessary to achieve a similar reduction in the conventional glass microfibre separator. But such thin material should have improved mechanical properties and can make the battery more prone to develop short circuits across the separator. In order to avoid this problem, a new microporous polyethylene membrane has been developed and tested, with excellent mechanical properties, high porosity and low pore size. For these reasons, the final separator configuration includes a combination of both materials, improved non-woven glass microfibre and the polyethylene membrane. Batteries are designed and assembled by Tudor (Exide Technologies) as battery manufacturer and will be tested in real conditions by MGE UPS Systems as end user. Daramic for the membrane and Bernard Dumas for the glass microfibre mat, have developed and supplied the separators, while the Inorganic Chemistry Department of Córdoba University carries out fundamental research studies on very thin electrodes.

Corrosion management of PbCaSn alloys in lead-acid batteries: Effect of composition, metallographic state and voltage conditions

NASA Astrophysics Data System (ADS)

Rocca, E.; Bourguignon, G.; Steinmetz, J.

Since several years, lead calcium-based alloys have supplanted lead antimony alloys as structural materials for positive grids of lead-acid batteries in many applications, especially for VRLA batteries. Nevertheless, the positive grid corrosion probably remains one of the causes of rapid and premature failure of lead-acid batteries. The objective of the present study is to present a comprehensive study of the PbCaSn alloy corrosion in function of their composition, metallographic state and voltage conditions (discharge, overcharge, floating and cycling conditions). For that, four alloys PbCaSn x wt.% (x = 0, 0.6, 1.2, 2) were synthesized in two extreme metallurgical conditions and tested by four electrochemical lab-tests. Weight loss measurements and analyses by SEM, EPMA and XRD allowed to monitor the oxidation tests and to characterize the corrosion layers after the oxidation tests. The results show that the tin level in PbCaSn alloys should be adapted on the calcium concentration and the rate of overageing process, to maintain the beneficial effect of tin in service during the battery lifetime. According to our results, a Sn/Ca ratio of 2.5 gives good corrosion resistance in all potential conditions. Nevertheless, when tin level is too high, the corrosion layers can peel off from the metal, which involves a lack of cohesion between the collector and the paste, in cycling conditions. The anodic potential undergone by the metal is a second main factor determining the corrosion, especially the floating conditions and the frequency of deep discharge and overcharge. Thus the adjustment of the charge controller parameters of a battery system is a necessity to increase the lifetime of the grids and maintain a good rechargeability.

Multiple pass gas absorption cell utilizing a spherical mirror opposite one or more pair of obliquely disposed flat mirrors

NASA Technical Reports Server (NTRS)

Pearson, Richard (Inventor); Lynch, Dana H. (Inventor); Gunter, William D. (Inventor)

1995-01-01

A method and apparatus for passing light bundles through a multiple pass sampling cell is disclosed. The multiple pass sampling cell includes a sampling chamber having first and second ends positioned along a longitudinal axis of the sampling cell. The sampling cell further includes an entrance opening, located adjacent the first end of the sampling cell at a first azimuthal angular position. The entrance opening permits a light bundle to pass into the sampling cell. The sampling cell also includes an exit opening at a second azimuthal angular position. The light exit permits a light bundle to pass out of the sampling cell after the light bundle has followed a predetermined path.

The endothelial sample size analysis in corneal specular microscopy clinical examinations.

PubMed

Abib, Fernando C; Holzchuh, Ricardo; Schaefer, Artur; Schaefer, Tania; Godois, Ronialci

2012-05-01

To evaluate endothelial cell sample size and statistical error in corneal specular microscopy (CSM) examinations. One hundred twenty examinations were conducted with 4 types of corneal specular microscopes: 30 with each BioOptics, CSO, Konan, and Topcon corneal specular microscopes. All endothelial image data were analyzed by respective instrument software and also by the Cells Analyzer software with a method developed in our lab. A reliability degree (RD) of 95% and a relative error (RE) of 0.05 were used as cut-off values to analyze images of the counted endothelial cells called samples. The sample size mean was the number of cells evaluated on the images obtained with each device. Only examinations with RE < 0.05 were considered statistically correct and suitable for comparisons with future examinations. The Cells Analyzer software was used to calculate the RE and customized sample size for all examinations. Bio-Optics: sample size, 97 ± 22 cells; RE, 6.52 ± 0.86; only 10% of the examinations had sufficient endothelial cell quantity (RE < 0.05); customized sample size, 162 ± 34 cells. CSO: sample size, 110 ± 20 cells; RE, 5.98 ± 0.98; only 16.6% of the examinations had sufficient endothelial cell quantity (RE < 0.05); customized sample size, 157 ± 45 cells. Konan: sample size, 80 ± 27 cells; RE, 10.6 ± 3.67; none of the examinations had sufficient endothelial cell quantity (RE > 0.05); customized sample size, 336 ± 131 cells. Topcon: sample size, 87 ± 17 cells; RE, 10.1 ± 2.52; none of the examinations had sufficient endothelial cell quantity (RE > 0.05); customized sample size, 382 ± 159 cells. A very high number of CSM examinations had sample errors based on Cells Analyzer software. The endothelial sample size (examinations) needs to include more cells to be reliable and reproducible. The Cells Analyzer tutorial routine will be useful for CSM examination reliability and reproducibility.

Cell block samples from malignant pleural effusion might be valid alternative samples for anaplastic lymphoma kinase detection in patients with advanced non-small-cell lung cancer.

PubMed

Zhou, Jianya; Yao, Hongtian; Zhao, Jing; Zhang, Shumeng; You, Qihan; Sun, Ke; Zou, Yinying; Zhou, Caicun; Zhou, Jianying

2015-06-01

To evaluate the clinical value of cell block samples from malignant pleural effusion (MPE) as alternative samples to tumour tissue for anaplastic lymphoma kinase (ALK) detection in patients with advanced non-small-cell lung cancer (NSCLC). Fifty-two matched samples were eligible for analysis. ALK status was detected by Ventana immunohistochemistry (IHC) (with the D5F3 clone), reverse transcription polymerase chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH) in MPE cell block samples, and by FISH in tumour tissue block samples. In total, ALK FISH results were obtained for 52 tumour tissue samples and 41 MPE cell block samples. Eight cases (15.4%) were ALK-positive in tumour tissue samples by FISH, and among matched MPE cell block samples, five were ALK-positive by FISH, seven were ALK-positive by RT-PCR, and eight were ALK-positive by Ventana IHC. The ALK status concordance rates between tumour tissue and MPE cell block samples were 78.9% by FISH, 98.1% by RT-PCR, and 100% by Ventana IHC. In MPE cell block samples, the sensitivity and specificity of Ventana IHC (100% and 100%) and RT-PCR (87.5% and 100%) were higher than those of FISH (62.5% and 100%). Malignant pleural effusion cell block samples had a diagnostic performance for ALK detection in advanced NSCLC that was comparable to that of tumour tissue samples. MPE cell block samples might be valid alternative samples for ALK detection when tissue is not available. Ventana IHC could be the most suitable method for ALK detection in MPE cell block samples. © 2014 John Wiley & Sons Ltd.

Combinatorial Screening Of Inorganic And Organometallic Materials

DOEpatents

Li, Yi , Li, Jing , Britton, Ted W.

2002-06-25

A method for differentiating and enumerating nucleated red blood cells in a blood sample is described. The method includes the steps of lysing red blood cells of a blood sample with a lytic reagent, measuring nucleated blood cells by DC impedance measurement in a non-focused flow aperture, differentiating nucleated red blood cells from other cell types, and reporting nucleated red blood cells in the blood sample. The method further includes subtracting nucleated red blood cells and other interference materials from the count of remaining blood cells, and reporting a corrected white blood cell count of the blood sample. Additionally, the method further includes measuring spectrophotometric absorbance of the sample mixture at a predetermined wavelength of a hemoglobin chromogen formed upon lysing the blood sample, and reporting hemoglobin concentration of the blood sample.

Drug loaded silica coated MnFe2O4 ferromagnetic biomaterials for targeted cancer treatment

NASA Astrophysics Data System (ADS)

Anand, Vikas; Singh, K. J.; Kaur, Kulwinder; Bhatia, Gaurav

2017-05-01

Magnetically attracted silica coated MnFe2O4 samples have been prepared by using co-precipitation method. Structural changes have been confirmed from XRD spectra. Ferromagnetic behavior of samples has been studied by using vibration sample magnetometer. Cytotoxicity and cell culture of samples have been investigated by using human MG63 cell line and found that sample provide a healthy environment to the growth of cell lines. Drug carrier ability of sample has been checked with gentamycin as an antibiotic and results show that sample can be used as excellent drug carriers. Drug loaded samples can be easily targeted to specific area due to their attractive nature towards external magnetic field. Moreover, magnetic nanoparticles can also be used to kill the cancer cells by using hyperthermia technique. Hyperthermia is a process to raise the temperature of surrounding cells in the presence of external AC magnetic field above the maximum temperature limit for surviving of the cancer cells. Cancer cell can survive only up to 47°C. After that cancer cells start to die, but healthy cells can easily survive up to higher temperature. Our results indicate that prepared samples possess good drug carrier ability and hence, can be potential candidates for cancer cell treatment.

Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization.

PubMed

Nejlund, Sarah; Smith, Julie; Kraan, Jaco; Stender, Henrik; Van, Mai N; Langkjer, Sven T; Nielsen, Mikkel T; Sölétormos, György; Hillig, Thore

2016-08-01

A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of existing and upcoming cancer biomarkers. Blood samples from healthy volunteers were spiked with high (∼500) and low (∼50) number of tumor cells from culture. The samples were stored at -80C with cryopreservative dimethyl sulfoxide mixed with Roswell Park Memorial Institute 1640 medium. Flow cytometry tested if cryopreservation affected specific biomarkers regularly used to detect CTCs, i.e. cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) and white blood cell specific lymphocyte common antigen (CD45). After various time intervals (up to 6 months), samples were thawed and tumor cell recovery (enumeration) was examined. Clinical samples may differ from cell line studies, so the cryopreservation protocol was tested on 17 patients with invasive breast cancer and tumor cell recovery was examined. Two blood samples were drawn from each patient. Biomarkers, CK, CD45, and EpCAM, were not affected by the freezing and thawing procedures. Cryopreserved samples (n = 2) spiked with a high number of tumor cells (∼500) had a ∼90% recovery compared with the spiked fresh samples. In samples spiked with lower numbers of tumor cells (median = 43 in n = 5 samples), the recovery was 63% after cryopreservation (median 27 tumor cells), p = 0.03. With an even lower number of spiked tumor cells (median = 3 in n = 8 samples), the recovery rate of tumor cells after cryopreservation did not seem to be affected (median = 8), p = 0.09. Time of cryopreservation did not affect recovery. When testing the effect of cryopreservation on enumeration in clinical samples, no difference was observed in the number of CTCs between the fresh and the cryopreserved samples based on n = 17 pairs, p = 0.83; however, the variation was large. This large variation was confirmed by clinically paired fresh samples (n = 64 pairs), where 95% of the samples (<30 CTCs) vary in number up to ±15 CTCs, p = 0.18. A small loss of CTCs after cryopreservation may be expected; however, cryopreservation of CTCs for biomarker characterization for clinical applications seems promising.

Utility of Cytospin and Cell block Technology in Evaluation of Body Fluids and Urine Samples: A Comparative Study.

PubMed

Qamar, Irmeen; Rehman, Suhailur; Mehdi, Ghazala; Maheshwari, Veena; Ansari, Hena A; Chauhan, Sunanda

2018-01-01

Cytologic examination of body fluids commonly involves the use of direct or sediment smears, cytocentrifuge preparations, membrane filter preparations, or cell block sections. Cytospin and cell block techniques are extremely useful in improving cell yield of thin serous effusions and urine samples, and ensure high diagnostic efficacy. We studied cytospin preparations and cell block sections prepared from 180 samples of body fluids and urine samples to compare the relative efficiency of cell retrieval, preservation of cell morphology, ease of application of special stains, and diagnostic efficacy. Samples were collected and processed to prepare cytospin smears and cell block sections. We observed that overall, cell yield and preservation of individual cell morphology were better in cytospin preparations as compared to cell blocks, while preservation of architectural pattern was better in cell block sections. The number of suspicious cases also decreased on cell block sections, with increased detection of malignancy. It was difficult to prepare cell blocks from urine samples due to low cellularity. Cytospin technology is a quick, efficient, and cost-effective method of increasing cell yield in hypocellular samples, with better preservation of cell morphology. Cell blocks are better prepared from high cellularity fluids; however, tissue architecture is better studied, with improved rate of diagnosis and decrease in ambiguous results. Numerous sections can be prepared from a small amount of material. Special stains and immunochemical stains can be easily applied to cell blocks. It also provides a source of archival material.

Detection of Irradiation Treatment of Foods Using DNA `Comet Assay'

NASA Astrophysics Data System (ADS)

Khan, Hasan M.; Delincée, Henry

1998-06-01

Microgel electrophoresis of single cells (DNA comet assay) has been investigated to detect irradiation treatment of some food samples. These samples of fresh and frozen rainbow trout, red lentil, gram and sliced almonds were irradiated to 1 or 2 kGy using 10 MeV electron beam from a linear accelerator. Rainbow trout samples yielded good results with samples irradiated to 1 or 2 kGy showing fragmentation of DNA and, therefore, longer comets with no intact cells. Unirradiated samples showed shorter comets with a significant number of intact cells. For rainbow trout stored in a freezer for 11 days the irradiated samples can still be discerned by electrophoresis from unirradiated samples, however, the unirradiated trouts also showed some longer comets besides some intact cells. Radiation treatment of red lentils can also be detected by this method, i.e. no intact cells in 1 or 2 kGy irradiated samples and shorter comets and some intact cells in unirradiated samples. However, the results for gram and sliced almond samples were not satisfactory since some intact DNA cells were observed in irradiated samples as well. Probably, incomplete lysis has led to these deviating results.

Measurement of wood/plant cell or composite material attributes with computer assisted tomography

DOEpatents

West, Darrell C.; Paulus, Michael J.; Tuskan, Gerald A.; Wimmer, Rupert

2004-06-08

A method for obtaining wood-cell attributes from cellulose containing samples includes the steps of radiating a cellulose containing sample with a beam of radiation. Radiation attenuation information is collected from radiation which passes through the sample. The source is rotated relative to the sample and the radiation and collecting steps repeated. A projected image of the sample is formed from the collected radiation attenuation information, the projected image including resolvable features of the cellulose containing sample. Cell wall thickness, cell diameter (length) and cell vacoule diameter can be determined. A system for obtaining physical measures from cellulose containing samples includes a radiation source, a radiation detector, and structure for rotating the source relative to said sample. The system forms an image of the sample from the radiation attenuation information, the image including resolvable features of the sample.

Utility of Cytospin and Cell block Technology in Evaluation of Body Fluids and Urine Samples: A Comparative Study

PubMed Central

Qamar, Irmeen; Rehman, Suhailur; Mehdi, Ghazala; Maheshwari, Veena; Ansari, Hena A.; Chauhan, Sunanda

2018-01-01

Background: Cytologic examination of body fluids commonly involves the use of direct or sediment smears, cytocentrifuge preparations, membrane filter preparations, or cell block sections. Cytospin and cell block techniques are extremely useful in improving cell yield of thin serous effusions and urine samples, and ensure high diagnostic efficacy. Materials and Methods: We studied cytospin preparations and cell block sections prepared from 180 samples of body fluids and urine samples to compare the relative efficiency of cell retrieval, preservation of cell morphology, ease of application of special stains, and diagnostic efficacy. Samples were collected and processed to prepare cytospin smears and cell block sections. Results: We observed that overall, cell yield and preservation of individual cell morphology were better in cytospin preparations as compared to cell blocks, while preservation of architectural pattern was better in cell block sections. The number of suspicious cases also decreased on cell block sections, with increased detection of malignancy. It was difficult to prepare cell blocks from urine samples due to low cellularity. Conclusions: Cytospin technology is a quick, efficient, and cost-effective method of increasing cell yield in hypocellular samples, with better preservation of cell morphology. Cell blocks are better prepared from high cellularity fluids; however, tissue architecture is better studied, with improved rate of diagnosis and decrease in ambiguous results. Numerous sections can be prepared from a small amount of material. Special stains and immunochemical stains can be easily applied to cell blocks. It also provides a source of archival material. PMID:29643653

Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function.

PubMed

Germann, Anja; Oh, Young-Joo; Schmidt, Tomm; Schön, Uwe; Zimmermann, Heiko; von Briesen, Hagen

2013-10-01

The ability to analyze cryopreserved peripheral blood mononuclear cell (PBMC) from biobanks for antigen-specific immunity is necessary to evaluate response to immune-based therapies. To ensure comparable assay results, collaborative research in multicenter trials needs reliable and reproducible cryopreservation that maintains cell viability and functionality. A standardized cryopreservation procedure is comprised of not only sample collection, preparation and freezing but also low temperature storage in liquid nitrogen without any temperature fluctuations, to avoid cell damage. Therefore, we have developed a storage approach to minimize suboptimal storage conditions in order to maximize cell viability, recovery and T-cell functionality. We compared the influence of repeated temperature fluctuations on cell health from sample storage, sample sorting and removal in comparison to sample storage without temperature rises. We found that cyclical temperature shifts during low temperature storage reduce cell viability, recovery and immune response against specific-antigens. We showed that samples handled under a protective hood system, to avoid or minimize such repeated temperature rises, have comparable cell viability and cell recovery rates to samples stored without any temperature fluctuations. Also T-cell functionality could be considerably increased with the use of the protective hood system compared to sample handling without such a protection system. This data suggests that the impact of temperature fluctuation on cell integrity should be carefully considered in future clinical vaccine trials and consideration should be given to optimal sample storage conditions. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Biological evaluation of partially stabilized zirconia added HA/HDPE composites with osteoblast and fibroblast cell lines.

PubMed

Yari Sadi, Amir; Shokrgozar, Mohammad Ali; Homaeigohar, Seyed Shahin; Khavandi, Alireza

2008-06-01

In the present study, the biocompatibility of partially stabilized zirconia (PSZ) added hydroxyapatite (HA)--high density polyethylene (HDPE) composites was evaluated by proliferation and cell attachment assays on two osteoblast cell lines (G-292, Saos-2) and a type of fibroblast cell isolated from bone tissue namely HBF in different time intervals. Cell-material interactions on the surface of the composites were observed by scanning electron microscopy (SEM). The effect of composites on the behavior of osteoblast and fibroblast cells was compared with those of HDPE and Tissue Culture Poly Styrene (TPS) (as negative control) samples. Results showed that the composite samples supported a higher proliferation rate of osteoblast cells in the presence of composite samples as compared to the HDPE and TPS samples after 3, 7 and 14 days of incubation period. It was showed that an equal or in some cases an even higher proliferation rate of G-292 and Saos-2 osteoblast cells on composite samples in compare to negative controls in culture period (P < 0.05). The number of adhered cells on the composite samples was equal and in some cases higher than the number adhered on the HDPE and TPS samples after the above mentioned incubation periods (P < 0.05). Adhered cells presented a normal morphology by SEM and many of the cells were seen to be undergoing cell division.

Plural output optimetric sample cell and analysis system

NASA Technical Reports Server (NTRS)

Haley, F. C. (Inventor)

1971-01-01

An apparatus suitable for receiving a sample for optimetric analysis includes a sample cell comprising an opaque hollow tube. Several apertures are defined in the wall of the tubing and a lens barrel which extends beyond to opposite surfaces of the wall is supported within at least one of the apertures. A housing is provided with one channel for receiving the sample cell and a series of channels extending from the exterior housing to the sample cell apertures. A filter element is housed in each of these latter channels. These channels slidingly receive an excitation light source for a photodetector cell to permit selective focusing. A sample cell containing at least three apertures in the walls can be mounted for rotation relative to a light source or photoconduction means for simultaneous or alternative optimetric determination of the components of a single sample. The sample cell is fabricated by supporting a lens barrel within the aperture. A molten portion of glass is deposited in the lens barrel and cooled while in a horizontal position to form a lens having an acceptable angle.

Effect of Thermodiffusion Nitriding on Cytocompatibility of Ti-6Al-4V Titanium Alloy

NASA Astrophysics Data System (ADS)

Pohrelyuk, I. M.; Tkachuk, O. V.; Proskurnyak, R. V.; Boiko, N. M.; Kluchivska, O. Yu.; Stoika, R. S.

2016-04-01

The nitrided layer was formed on the surface of Ti-6Al-4V titanium alloy by the thermodiffusion saturation in nitrogen at the atmospheric pressure. The study of the vitality of pseudonormal human embryo kidney cells of the HEK293T line showed that their cultivation in the presence of the untreated alloy sample is accompanied by a statistically significant reduction in the number of living cells compared with the control sample (untreated cells), whereas their cultivation in the presence of the nitrided alloy sample does not change the cell number considerably. In addition, it was shown that cell behavior in the presence of the nitrided sample differs only slightly from the control sample, whereas the growth of cells in the presence of the untreated alloy differed significantly from that in the control sample, demonstrating small groups of cells instead of their big clusters.

Optimised cord blood sample selection for small‑scale CD34+ cell immunomagnetic isolation.

PubMed

Perdomo-Arciniegas, Ana-María; Vernot, Jean-Paul

2012-03-01

Haematopoietic stem cells (HSCs) are defined as multipotential cells, capable of self-renewal and reconstituting in vivo the haematopoietic compartment. The CD34 antigen is considered an important HSCs marker in humans. Immunomagnetic isolation, by targeting CD34 antigen, is widely used for human HSC separation. This method allows the enrichment of human HSCs that are present at low frequencies in umbilical cord blood (CB). Immunomagnetic CD34+-cell isolation reproducibility, regarding cell yield and purity, is affected by the CD34+ cell frequency and total cell numbers present in a given sample; CB HSC purification may thus yield variable results, which also depend on the volume and density fractionation-derived cell loss of a CB sample. The uncertainty of such an outcome and associated technical costs call for a cost-effective sample screening strategy. A correlation analysis using clinical and laboratory data from 59 CB samples was performed to establish predictive variables for CD34+-immunomagnetic HSCs isolation. This study described the positive association of CD34+-cell isolation with white and red cell numbers present after cell fractionation. Furthermore, purity has been correlated with lymphocyte percentages. Predictive variable cut-off values, which are particularly useful in situations involving low CB volumes being collected (such as prevalent late umbilical cord clamping clinical practice), were proposed for HSC isolation sampling. Using the simple and cost-effective CB sample screening criteria described here would lead to avoiding costly inefficient sample purification, thereby ensuring that pure CD34+ cells are obtained in the desired numbers following CD34 immunomagnetic isolation.

Nucleotide composition analysis of tRNA from leukemia patient cell samples and human cell lines.

PubMed Central

Agris, P F

1975-01-01

A technique developed for analysis of less than microgram quantities of tRNA has been applied to the study of human leukemia. Leucocytes from peripheal blood and bone marrow samples of six, untreated leukemia patients and cells of five different established human cell lines were maintained for 18 hours in media containing (32P)-phosphate. Incorporation of radioactive phosphate into the cells from the patient samples was slightly less than that of the cell lines. Likewise, incorporation of (32P)-phosphate into the tRNA of the patient samples (approximately 5 x 106 DPM/mug tRNA) was also less then that incorporated into the tRNA of the cell lines. The major and minor nucleotide compositions of the unfractionated tRNA preparations from each patient sample and each cell line were determined and compared. Similarities and differences in the major and minor nucleotide compositions of the tRNA preparations are discussed with reference to types of leukemia and the importance of patient sample analysis versus analysis of cultured human cells. PMID:1057159

Performance evaluation of the Abbott CELL-DYN Emerald for use as a bench-top analyzer in a research setting.

PubMed

Khoo, T-L; Xiros, N; Guan, F; Orellana, D; Holst, J; Joshua, D E; Rasko, J E J

2013-08-01

The CELL-DYN Emerald is a compact bench-top hematology analyzer that can be used for a three-part white cell differential analysis. To determine its utility for analysis of human and mouse samples, we evaluated this machine against the larger CELL-DYN Sapphire and Sysmex XT2000iV hematology analyzers. 120 human (normal and abnormal) and 30 mouse (normal and abnormal) samples were analyzed on both the CELL-DYN Emerald and CELL-DYN Sapphire or Sysmex XT2000iV analyzers. For mouse samples, the CELL-DYN Emerald analyzer required manual recalibration based on the histogram populations. Analysis of the CELL-DYN Emerald showed excellent precision, within accepted ranges (white cell count CV% = 2.09%; hemoglobin CV% = 1.68%; platelets CV% = 4.13%). Linearity was excellent (R² ≥ 0.99), carryover was minimal (<1%), and overall interinstrument agreement was acceptable for both human and mouse samples. Comparison between the CELL-DYN Emerald and Sapphire analyzers for human samples or Sysmex XT2000iV analyzer for mouse samples showed excellent correlation for all parameters. The CELL-DYN Emerald was generally comparable to the larger reference analyzer for both human and mouse samples. It would be suitable for use in satellite research laboratories or as a backup system in larger laboratories. © 2012 John Wiley & Sons Ltd.

Effects of sample treatments on genome recovery via single-cell genomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clingenpeel, Scott; Schwientek, Patrick; Hugenholtz, Philip

2014-06-13

It is known that single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we demonstrate that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.

Evaluation of Buccal Cell Samples for Studies of Oral Microbiota.

PubMed

Yu, Guoqin; Phillips, Steve; Gail, Mitchell H; Goedert, James J; Humphrys, Michael; Ravel, Jacques; Ren, Yanfang; Caporaso, Neil E

2017-02-01

The human microbiota is postulated to affect cancer risk, but collecting microbiota specimens with prospective follow-up for diseases will take time. Buccal cell samples have been obtained from mouthwash for the study of human genomic DNA in many cohort studies. Here, we evaluate the feasibility of using buccal cell samples to examine associations of human microbiota and disease risk. We obtained buccal cells from mouthwash in 41 healthy participants using a protocol that is widely employed to obtain buccal cells for the study of human DNA. We compared oral microbiota from buccal cells with that from eight other oral sample types collected by following the protocols of the Human Microbiome Project. Microbiota profiles were determined by sequencing 16S rRNA gene V3-V4 region. Compared with each of the eight other oral samples, the buccal cell samples had significantly more observed species (P < 0.002) and higher alpha diversity (Shannon index, P < 0.02). The microbial communities were more similar (smaller beta diversity) among buccal cells samples than in the other samples (P < 0.001 for 12 of 16 weighted and unweighted UniFrac distance comparisons). Buccal cell microbial profiles closely resembled saliva but were distinct from dental plaque and tongue dorsum. Stored buccal cell samples in prospective cohort studies are a promising resource to study associations of oral microbiota with disease. The feasibility of using existing buccal cell collections in large prospective cohorts allows investigations of the role of oral microbiota in chronic disease etiology in large population studies possible today. Cancer Epidemiol Biomarkers Prev; 26(2); 249-53. ©2016 AACR. ©2016 American Association for Cancer Research.

Infectious hematopoietic necrosis virus detected by separation and incubation of cells from salmonid cavity fluid.

USGS Publications Warehouse

Mulcahy, D.; Batts, W.N.

1987-01-01

Infectious hematopoietic necrosis (IHN) virus is usually detected by inoculating susceptible cell cultures with cavity ("ovarian") fluid (CF) from spawning females. We identified additional adult carriers of virus in spawning populations of steelhead trout (Salmo gairdneri) and sockeye salmon (Oncorhynchus nerka) by collecting nonerythrocytic cells from CF samples by low-speed centrifugation, culturing the cells for at least 7 d at 15 °C, and then testing the culture medium for virus. Virus appeared in the cultured cells from some samples of CF that remained negative during incubation. In additional samples of CF from these species, the virus titer increased in cultured cells compared with the titer in the original CF sample. With chinook salmon (O.tshawytscha), no negative samples converted to positive during incubation, but the virus titer was retained in incubated CF cells, but not in cell-free CF.

Composition, indigenous proteolytic enzymes and coagulating behaviour of ewe milk as affected by somatic cell count.

PubMed

Albenzio, Marzia; Santillo, Antonella; Caroprese, Mariangela; Schena, Laura; Russo, Donatella Esterina; Sevi, Agostino

2011-11-01

This study was undertaken to assess the effect of somatic cell count in ewe milk on i) composition and hygienic traits; ii) plasmin, cathepsin and elastase activities; iii) leukocyte differential count; iv) renneting parameters. Individual ewe milk samples were grouped according to somatic cell count (SCC) into five classes: SC300 (<300 000 cells/ml), SC500 (from 301 000 to 500 000 cells/ml), SC1000 (from 501 000 to 1 000 000 cells/ml), SC2000 (from 1 001 000 to 2 000 000 cells/ml) and SC>2000 (>2 001 000 cells/ml). Individual milk samples were analysed for pH, chemical composition, microbial features, indigenous proteolytic enzymes, differential leukocyte population, and renneting parameters. Milk yield, lactose, protein, non casein nitrogen, microbial features were affected by SCC level. Plasmin and elastase activities were the highest in samples with more than 1 000 000 cells/ml; plasmin had intermediate values in samples with 300 000 to 1 000 000 cells/ml and the lowest in samples with less than 300 000 cells/ml of milk. Cathepsin D showed significantly lower values in SC300 and SC1000 classes than in SC500, SC2000 and SC>2000 classes. The highest percentages of lymphocyte were found in samples with less than 1 000 000 cells/ml, while the highest levels of polymorphonuclear leukocyte were found in samples with more than 1 000 000 cells/ml of milk. Longer clotting time was found in SC>2000 samples, while reduced clot firmness was observed in SC500 and SC>2000 samples. Results on milk yield and on compositional parameters evidenced an impairment of udder efficiency in ewe milk samples starting from 300 000 cells/ml. Plasmin activity in milk can be considered as a marker of the synthetic and secreting ability of the mammary gland; furthermore plasmin and elastase were consistent with the health status of the udder. Finally cathepsin D played a role in the worsening of renneting properties of ewe milk.

Detection of sepsis in patient blood samples using CD64 expression in a microfluidic cell separation device.

PubMed

Zhang, Ye; Li, Wenjie; Zhou, Yun; Johnson, Amanda; Venable, Amanda; Hassan, Ahmed; Griswold, John; Pappas, Dimitri

2017-12-18

A microfluidic affinity separation device was developed for the detection of sepsis in critical care patients. An affinity capture method was developed to capture cells based on changes in CD64 expression in a single, simple microfluidic chip for sepsis detection. Both sepsis patient samples and a laboratory CD64+ expression model were used to validate the microfluidic assay. Flow cytometry analysis showed that the chip cell capture had a linear relationship with CD64 expression in laboratory models. The Sepsis Chip detected an increase in upregulated neutrophil-like cells when the upregulated cell population is as low as 10% of total cells spiked into commercially available aseptic blood samples. In a proof of concept study, blood samples obtained from sepsis patients within 24 hours of diagnosis were tested on the chip to further validate its performance. On-chip CD64+ cell capture from 10 patient samples (619 ± 340 cells per chip) was significantly different from control samples (32 ± 11 cells per chip) and healthy volunteer samples (228 ± 95 cells per chip). In addition, the on-chip cell capture has a linear relationship with CD64 expression indicating our approach can be used to measure CD64 expression based on total cell capture on Sepsis Chip. Our method has proven to be sensitive, accurate, rapid, and cost-effective. Therefore, this device is a promising detection platform for neutrophil activation and sepsis diagnosis.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion.

PubMed

Kasurinen, Stefanie; Happo, Mikko S; Rönkkö, Teemu J; Orasche, Jürgen; Jokiniemi, Jorma; Kortelainen, Miika; Tissari, Jarkko; Zimmermann, Ralf; Hirvonen, Maija-Riitta; Jalava, Pasi I

2018-01-01

In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity.

Characteristics of HIV target CD4 T cells collected using different sampling methods from the genital tract of HIV seronegative women.

PubMed

Iyer, Smita S; Sabula, Michael J; Mehta, C Christina; Haddad, Lisa B; Brown, Nakita L; Amara, Rama R; Ofotokun, Igho; Sheth, Anandi N

2017-01-01

Understanding the immune profile of CD4 T cells, the primary targets for HIV, in the female genital tract (FGT) is critical for evaluating and developing effective biomedical HIV prevention strategies in women. However, longitudinal investigation of HIV susceptibility markers expressed by FGT CD4 T cells has been hindered by low cellular yield and risk of sampling-associated trauma. We investigated three minimally invasive FGT sampling methods to characterize and compare CD4 T cell yield and phenotype with the goal of establishing feasible sampling strategies for immune profiling of mucosal CD4 T cells. FGT samples were collected bimonthly from 12 healthy HIV negative women of reproductive age in the following order: 1) Cervicovaginal lavage (CVL), 2) two sequential endocervical flocked swabs (FS), and 3) two sequential endocervical cytobrushes (CB1, CB2). Cells were isolated and phentoyped via flow cytometry. CD4 T cell recovery was highest from each individual CB compared to either CVL or FS (p < 0.0001). The majority of CD4 T cells within the FGT, regardless of sampling method, expressed CCR5 relative to peripheral blood (p < 0.01). Within the CB, CCR5+ CD4 T cells expressed significantly higher levels of α4β7, CD69, and low levels of CD27 relative to CCR5- CD4 T cells (all p < 0.001). We also identified CD4 Treg lineage cells expressing CCR5 among CB samples. Using three different mucosal sampling methods collected longitudinally we demonstrate that CD4 T cells within the FGT express CCR5 and α4β7 and are highly activated, attributes which could act in concert to facilitate HIV acquisition. FS and CB sampling methods can allow for investigation of strategies to reduce HIV target cells in the FGT and could inform the design and interpretation microbicide and vaccine studies in women.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion

PubMed Central

Happo, Mikko S.; Rönkkö, Teemu J.; Orasche, Jürgen; Jokiniemi, Jorma; Kortelainen, Miika; Tissari, Jarkko; Zimmermann, Ralf; Hirvonen, Maija-Riitta; Jalava, Pasi I.

2018-01-01

Background In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. Methods We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. Results We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. Conclusions All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity. PMID:29466392

[Susceptibility of induced sickle in samples of heterozygous hemoglobin S patients (sickle cell trait) suffering diabetes mellitus type 2].

PubMed

Díaz-Piedra, Pablo; Cervantes-Villagrana, Alberto Rafael; Ramos-Jiménez, Raúl; Presno-Bernal, José Miguel; Cervantes-Villagrana, Rodolfo Daniel

2015-01-01

Hemoglobin S is an abnormal protein that induces morphological changes in erythrocyte in low-oxygen conditions. In Mexico, it is reported that up to 13.7% of the population with mutation in one allele are considered asymptomatic (sickle cell trait). The sickle cell trait and diabetes mellitus are conditions that occur together in more than one million patients worldwide. Both diseases possibly produce microvascular changes in retinopathy and acute chest syndrome. The aim of this study was to evaluate the induction of sickle cells in samples of diabetic patients with sickle cell trait to identify altered red cell parameters. We obtained samples of diabetic patients to determine hemoglobin A1c and S; furthermore, red blood cell biometrics data were analyzed. We found that older men with diabetes were susceptible to generate sickle cells and this correlated with reduced red blood cell count and an increase in media cell volume. In samples of women diabetes, there were no differences. We conclude that samples from patients with sickle cell trait and diabetes can cause sickle cells with high frequency in men, with lower red blood cells count and increased mean corpuscular volume as susceptibility parameters.

Use of immunomagnetic separation for the detection of Desulfovibrio vulgaris from environmental samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, R.; Hazen, T.C.; Joyner, D.C.

2011-04-15

Immunomagnetic separation (IMS) has proved highly efficient for recovering microorganisms from heterogeneous samples. Current investigation targeted the separation of viable cells of the sulfate-reducing bacterium, Desulfovibrio vulgaris. Streptavidin-coupled paramagnetic beads and biotin labeled antibodies raised against surface antigens of this microorganism were used to capture D. vulgaris cells in both bioreactor grown laboratory samples and from extremely low-biomass environmental soil and subsurface drilling samples. Initial studies on detection, recovery efficiency and viability for IMS were performed with laboratory grown D. vulgaris cells using various cell densities. Efficiency of cell isolation and recovery (i.e., release of the microbial cells from themore » beads following separation) was followed by microscopic imaging and acridine orange direct counts (AODC). Excellent recovery efficiency encouraged the use of IMS to capture Desulfovibrio spp. cells from low-biomass environmental samples. The environmental samples were obtained from a radionuclide-contaminated site in Germany and the chromium (VI)-contaminated Hanford site, an ongoing bioremediation project of the U.S. Department of Energy. Field deployable IMS technology may greatly facilitate environmental sampling and bioremediation process monitoring and enable transcriptomics and proteomics/metabolomics-based studies directly on cells collected from the field.« less

Development of a novel cell sorting method that samples population diversity in flow cytometry.

PubMed

Osborne, Geoffrey W; Andersen, Stacey B; Battye, Francis L

2015-11-01

Flow cytometry based electrostatic cell sorting is an important tool in the separation of cell populations. Existing instruments can sort single cells into multi-well collection plates, and keep track of cell of origin and sorted well location. However currently single sorted cell results reflect the population distribution and fail to capture the population diversity. Software was designed that implements a novel sorting approach, "Slice and Dice Sorting," that links a graphical representation of a multi-well plate to logic that ensures that single cells are sampled and sorted from all areas defined by the sort region/s. Therefore the diversity of the total population is captured, and the more frequently occurring or rarer cell types are all sampled. The sorting approach was tested computationally, and using functional cell based assays. Computationally we demonstrate that conventional single cell sorting can sample as little as 50% of the population diversity dependant on the population distribution, and that Slice and Dice sorting samples much more of the variety present within a cell population. We then show by sorting single cells into wells using the Slice and Dice sorting method that there are cells sorted using this method that would be either rarely sorted, or not sorted at all using conventional single cell sorting approaches. The present study demonstrates a novel single cell sorting method that samples much more of the population diversity than current methods. It has implications in clonal selection, stem cell sorting, single cell sequencing and any areas where population heterogeneity is of importance. © 2015 International Society for Advancement of Cytometry.

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring

PubMed Central

Cao, Yuhong; Chen, Haodong; Birey, Fikri; Leal-Ortiz, Sergio A.; Han, Crystal M.; Santiago, Juan G.; Paşca, Sergiu P.; Wu, Joseph C.; Melosh, Nicholas A.

2017-01-01

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation. PMID:28223521

Interferometrically stable, enclosed, spinning sample cell for spectroscopic experiments on air-sensitive samples

NASA Astrophysics Data System (ADS)

Baranov, Dmitry; Hill, Robert J.; Ryu, Jisu; Park, Samuel D.; Huerta-Viga, Adriana; Carollo, Alexa R.; Jonas, David M.

2017-01-01

In experiments with high photon flux, it is necessary to rapidly remove the sample from the beam and to delay re-excitation until the sample has returned to equilibrium. Rapid and complete sample exchange has been a challenge for air-sensitive samples and for vibration-sensitive experiments. Here, a compact spinning sample cell for air and moisture sensitive liquid and thin film samples is described. The principal parts of the cell are a copper gasket sealed enclosure, a 2.5 in. hard disk drive motor, and a reusable, chemically inert glass sandwich cell. The enclosure provides an oxygen and water free environment at the 1 ppm level, as demonstrated by multi-day tests with sodium benzophenone ketyl radical. Inside the enclosure, the glass sandwich cell spins at ≈70 Hz to generate tangential speeds of 7-12 m/s that enable complete sample exchange at 100 kHz repetition rates. The spinning cell is acoustically silent and compatible with a ±1 nm rms displacement stability interferometer. In order to enable the use of the spinning cell, we discuss centrifugation and how to prevent it, introduce the cycle-averaged resampling rate to characterize repetitive excitation, and develop a figure of merit for a long-lived photoproduct buildup.

Interferometrically stable, enclosed, spinning sample cell for spectroscopic experiments on air-sensitive samples.

PubMed

Baranov, Dmitry; Hill, Robert J; Ryu, Jisu; Park, Samuel D; Huerta-Viga, Adriana; Carollo, Alexa R; Jonas, David M

2017-01-01

In experiments with high photon flux, it is necessary to rapidly remove the sample from the beam and to delay re-excitation until the sample has returned to equilibrium. Rapid and complete sample exchange has been a challenge for air-sensitive samples and for vibration-sensitive experiments. Here, a compact spinning sample cell for air and moisture sensitive liquid and thin film samples is described. The principal parts of the cell are a copper gasket sealed enclosure, a 2.5 in. hard disk drive motor, and a reusable, chemically inert glass sandwich cell. The enclosure provides an oxygen and water free environment at the 1 ppm level, as demonstrated by multi-day tests with sodium benzophenone ketyl radical. Inside the enclosure, the glass sandwich cell spins at ≈70 Hz to generate tangential speeds of 7-12 m/s that enable complete sample exchange at 100 kHz repetition rates. The spinning cell is acoustically silent and compatible with a ±1 nm rms displacement stability interferometer. In order to enable the use of the spinning cell, we discuss centrifugation and how to prevent it, introduce the cycle-averaged resampling rate to characterize repetitive excitation, and develop a figure of merit for a long-lived photoproduct buildup.

Palladium-based Mass-Tag Cell Barcoding with a Doublet-Filtering Scheme and Single Cell Deconvolution Algorithm

PubMed Central

Zunder, Eli R.; Finck, Rachel; Behbehani, Gregory K.; Amir, El-ad D.; Krishnaswamy, Smita; Gonzalez, Veronica D.; Lorang, Cynthia G.; Bjornson, Zach; Spitzer, Matthew H.; Bodenmiller, Bernd; Fantl, Wendy J.; Pe’er, Dana; Nolan, Garry P.

2015-01-01

SUMMARY Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption, and shortens instrument measurement time. Here, we present an optimized MCB protocol with several improvements over previously described methods. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3–4 h, not including sample acquisition time of ~1 h per million cells. PMID:25612231

21 CFR 660.36 - Samples and protocols.

Code of Federal Regulations, 2014 CFR

2014-04-01

..., whenever a new donor is used, a sample of red blood cells from each new donor used in a cell panel intended... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.36 Samples... distribution of each lot of Reagent Red Blood Cells for detection or identification of unexpected antibodies...

21 CFR 660.36 - Samples and protocols.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., whenever a new donor is used, a sample of red blood cells from each new donor used in a cell panel intended... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.36 Samples... distribution of each lot of Reagent Red Blood Cells for detection or identification of unexpected antibodies...

21 CFR 660.36 - Samples and protocols.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., whenever a new donor is used, a sample of red blood cells from each new donor used in a cell panel intended... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.36 Samples... distribution of each lot of Reagent Red Blood Cells for detection or identification of unexpected antibodies...

MC3T3-E1 cell response to stainless steel 316L with different surface treatments.

PubMed

Zhang, Hongyu; Han, Jianmin; Sun, Yulong; Huang, Yongling; Zhou, Ming

2015-11-01

In the present study, stainless steel 316L samples with polishing, aluminum oxide blasting, and hydroxyapatite (HA) coating were prepared and characterized through a scanning electron microscope (SEM), optical interferometer (surface roughness, Sq), contact angle, surface composition and phase composition analyses. Osteoblast-like MC3T3-E1 cell adhesion on the samples was investigated by cell morphology using a SEM (4h, 1d, 3d, 7d), and cell proliferation was assessed by MTT method at 1d, 3d, and 7d. In addition, adsorption of bovine serum albumin on the samples was evaluated at 1h. The polished sample was smooth (Sq: 1.8nm), and the blasted and HA coated samples were much rougher (Sq: 3.2μm and 7.8μm). Within 1d of incubation, the HA coated samples showed the best cell morphology (e.g., flattened shape and complete spread), but there was no significant difference after 3d and 7d of incubation for all the samples. The absorbance value for the HA coated samples was the highest after 1d and 3d of incubation, indicating better cell viability. However, it reduced to the lowest value at 7d. Protein adsorption on the HA coated samples was the highest at 1h. The results indicate that rough stainless steel surface improves cell adhesion and morphology, and HA coating contributes to superior cell adhesion, but inhibits cell proliferation. Copyright © 2015. Published by Elsevier B.V.

A mechanical cell disruption microfluidic platform based on an on-chip micropump.

PubMed

Cheng, Yinuo; Wang, Yue; Wang, Zhiyuan; Huang, Liang; Bi, Mingzhao; Xu, Wenxiao; Wang, Wenhui; Ye, Xiongying

2017-03-01

Cell disruption plays a vital role in detection of intracellular components which contain information about genetic and disease characteristics. In this paper, we demonstrate a novel microfluidic platform based on an on-chip micropump for mechanical cell disruption and sample transport. A 50  μ l cell sample can be effectively lysed through on-chip multi-disruption in 36 s without introducing any chemical agent and suffering from clogging by cellular debris. After 30 cycles of circulating disruption, 80.6% and 90.5% cell disruption rates were achieved for the HEK293 cell sample and human natural killer cell sample, respectively. Profiting from the feature of pump-on-chip, the highly integrated platform enables more convenient and cost-effective cell disruption for the analysis of intracellular components.

A mechanical cell disruption microfluidic platform based on an on-chip micropump

PubMed Central

Cheng, Yinuo; Wang, Yue; Wang, Zhiyuan; Bi, Mingzhao; Xu, Wenxiao; Ye, Xiongying

2017-01-01

Cell disruption plays a vital role in detection of intracellular components which contain information about genetic and disease characteristics. In this paper, we demonstrate a novel microfluidic platform based on an on-chip micropump for mechanical cell disruption and sample transport. A 50 μl cell sample can be effectively lysed through on-chip multi-disruption in 36 s without introducing any chemical agent and suffering from clogging by cellular debris. After 30 cycles of circulating disruption, 80.6% and 90.5% cell disruption rates were achieved for the HEK293 cell sample and human natural killer cell sample, respectively. Profiting from the feature of pump-on-chip, the highly integrated platform enables more convenient and cost-effective cell disruption for the analysis of intracellular components. PMID:28798848

Apparatus and Methods for Photoacoustic Measurement of Light Absorption of Particulate and Gaseous Species

NASA Technical Reports Server (NTRS)

Brown, William (Inventor); Yu, Zhenhong (Inventor); Kebabian, Paul L. (Inventor); Assif, James (Inventor)

2017-01-01

In one embodiment, a photoacoustic effect measurement instrument for measuring a species (e.g., a species of PM) in a gas employs a pair of differential acoustic cells including a sample cell that receives sample gas including the species, and a reference cell that receives a filtered version of the sample gas from which the species has been substantially removed. An excitation light source provides an amplitude modulated beam to each of the acoustic cells. An array of multiple microphones is mounted to each of the differential acoustic cells, and measures an acoustic wave generated in the respective acoustic cell by absorption of light by sample gas therein to produce a respective signal. The microphones are isolated from sample gas internal to the acoustic cell by a film. A preamplifier determines a differential signal and a controller calculates concentration of the species based on the differential signal.

Glycosyltransferases as marker genes for the quantitative polymerase chain reaction-based detection of circulating tumour cells from blood samples of patients with breast cancer undergoing adjuvant therapy.

PubMed

Kölbl, Alexandra C; Hiller, Roman A; Ilmer, Mathias; Liesche, Friederike; Heublein, Sabine; Schröder, Lennard; Hutter, Stefan; Friese, Klaus; Jeschke, Udo; Andergassen, Ulrich

2015-08-01

Altered glycosylation is a predominant feature of tumour cells; it serves for cell adhesion and detachment, respectively, and facilitates the immune escape of these cells. Therefore changes in the expression of glycosyltransferase genes could help to identify circulating tumour cells (CTCs) in the blood samples of cancer patients using a quantitative polymerase chain reaction (PCR) approach. Blood samples of healthy donors were inoculated with certain numbers of established breast cancer cell line cells, thus creating a model system. These samples were analysed by quantitative PCR for the expression of six different glycosyltransferase genes. The three genes with the best results in the model system were consecutively applied to samples from adjuvant breast cancer patients and of healthy donors. FUT3 and GALNT6 showed the highest increase in relative expression, while GALNT6 and ST3GAL3 were the first to reach statistically significant different ∆CT-values comparing the sample with and without addition of tumour cells. These three genes were applied to patient samples, but did not show any significant results that may suggest the presence of CTCs in the blood. Although the relative expression of some of the glycosyltransferase genes exhibited reasonable results in the model system, their application to breast cancer patient samples will have to be further improved, e.g. by co-analysis of patient blood samples by gold-standard methods.

New method for estimating bacterial cell abundances in natural samples by use of sublimation

NASA Technical Reports Server (NTRS)

Glavin, Daniel P.; Cleaves, H. James; Schubert, Michael; Aubrey, Andrew; Bada, Jeffrey L.

2004-01-01

We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples, including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert, were heated to a temperature of 500 degrees C for several seconds under reduced pressure. The sublimate was collected on a cold finger, and the amount of adenine released from the samples was then determined by high-performance liquid chromatography with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from approximately 10(5) to 10(9) E. coli cell equivalents per gram. For most of these samples, the sublimation-based cell counts were in agreement with total bacterial counts obtained by traditional DAPI (4,6-diamidino-2-phenylindole) staining.

Specimen Sample Preservation for Cell and Tissue Cultures

NASA Technical Reports Server (NTRS)

Meeker, Gabrielle; Ronzana, Karolyn; Schibner, Karen; Evans, Robert

1996-01-01

The era of the International Space Station with its longer duration missions will pose unique challenges to microgravity life sciences research. The Space Station Biological Research Project (SSBRP) is responsible for addressing these challenges and defining the science requirements necessary to conduct life science research on-board the International Space Station. Space Station will support a wide range of cell and tissue culture experiments for durations of 1 to 30 days. Space Shuttle flights to bring experimental samples back to Earth for analyses will only occur every 90 days. Therefore, samples may have to be retained for periods up to 60 days. This presents a new challenge in fresh specimen sample storage for cell biology. Fresh specimen samples are defined as samples that are preserved by means other than fixation and cryopreservation. The challenge of long-term storage of fresh specimen samples includes the need to suspend or inhibit proliferation and metabolism pending return to Earth-based laboratories. With this challenge being unique to space research, there have not been any ground based studies performed to address this issue. It was decided hy SSBRP that experiment support studies to address the following issues were needed: Fixative Solution Management; Media Storage Conditions; Fresh Specimen Sample Storage of Mammalian Cell/Tissue Cultures; Fresh Specimen Sample Storage of Plant Cell/Tissue Cultures; Fresh Specimen Sample Storage of Aquatic Cell/Tissue Cultures; and Fresh Specimen Sample Storage of Microbial Cell/Tissue Cultures. The objective of these studies was to derive a set of conditions and recommendations that can be used in a long duration microgravity environment such as Space Station that will permit extended storage of cell and tissue culture specimens in a state consistent with zero or minimal growth, while at the same time maintaining their stability and viability.

Polymerase chain reaction-based detection of B-cell monoclonality in cytologic specimens.

PubMed

Chen, Y T; Mercer, G O; Chen, Y

1993-11-01

Thirty-seven cytologic cell blocks were evaluated for B-cell monoclonality by polymerase chain reaction (PCR), 16 of them cytologically positive for lymphoma, and 21 suspicious for lymphoma but morphologically nondiagnostic. Of 37 specimens, 13 (35%) showed B-cell monoclonality, including six of 16 cytologically positive samples and seven of 21 cytologically suspicious ones. Of these 13 positive samples, seven were positive using crude lysates as substrates, and six additional positive samples were identified only when DNAs were purified and concentrated. Analysis of the DNAs further revealed poor polymerase chain reaction amplifiability and low DNA yield in many samples, indicating that cell block materials are suboptimal for this assay. We concluded that B-cell monoclonality can be detected in ethanol-fixed cytologic samples, and usage of unembedded material will likely improve the sensitivity. In specimens cytologically suspicious for lymphoma, polymerase chain reaction-based identification of monoclonal B-cell population supports the diagnosis of B-cell lymphoma and is a potentially useful test in solving this diagnostic dilemma.

Living cell manipulation, manageable sampling, and shotgun picoliter electrospray mass spectrometry for profiling metabolites.

PubMed

Gholipour, Yousef; Erra-Balsells, Rosa; Hiraoka, Kenzo; Nonami, Hiroshi

2013-02-01

A modified cell pressure probe and an online Orbitrap mass spectrometer were used to sample in situ plant single cells without any additional manipulation. The cell pressure probe, a quartz capillary tip filled with an oil mixture, was penetrated to various depths into parenchyma cells of tulip bulb scale, followed by a hydraulic continuity test to determine the exact location of the tip inside target cells. The operation was conducted under a digital microscope, and the capillary tip was photographed to calculate the volume of the cell sap sucked. The cell sap sample was then directly nebulized/ionized under high-voltage conditions at the entrance of the mass spectrometer. Several sugars, amino acids, organic acids, vitamins, fatty acids, and secondary metabolites were detected. Because picoliter solutions can be accurately handled and measured, known volumes of standard solutions can be added to cell sap samples inside the capillary tip to be used as references for metabolite characterization and relative quantitation. The high precision and sensitivity of the cell pressure probe and Orbitrap mass spectrometer allow for the manipulation and analysis of both femtoliter cell sap samples and standard solutions. Copyright © 2012 Elsevier Inc. All rights reserved.

Evaluation of Vitality in the Experimental Hanging Model of Rats by Using Immunohistochemical IL-1β Antibody Staining.

PubMed

Balandiz, Hüseyin; Pehlivan, Sultan; Çiçek, Ali Fuat; Tuğcu, Harun

2015-12-01

Hanging is the most common suicide method in the world, and the discrimination of antemortem-postmortem hanging must be done at autopsy. The aim of this experimental study was to examine the immunohistochemical expression of IL-1β antibody at the hanging mark skin samples of rats to discriminate antemortem and postmortem hangings. A total of 20 Wistar albino rats were used for this study. The groups were as follows: A-1, antemortem control group; A-2, antemortem second-hour hanging mark skin samples; A-3, antemortem 24th-hour hanging mark skin samples; A-4, antemortem 72nd-hour hanging mark skin samples; B-1, postmortem control group; and B-2, postmortem second-hour hanging mark skin samples. Interleukin-1β immunostaining was performed to all tissue samples. For epidermal cells, group A-1 samples did not show IL-1β immunostaining, group A-2 samples were severely immunostained, and groups A-3 and A-4 samples' staining were slightly decreased. There was no IL-1β antibody staining in groups B-1 and B-2 samples. For adnexal cells, groups A-1 and B-1 samples did not show IL-1β immunostaining, staining of group A-2 samples was mild to severe, and groups A-3 and A-4 samples' staining were slightly decreased. Half of the group B-2 samples did not show IL-1β immunostaining. For subepidermal cells, most of the samples of groups A-1 and B-1 showed slight immunostaining, groups A-2 and B-2 samples' staining were mild to severe, and there were slight immunostaining in groups A-3 and A-4 samples. The majority of vascular structure cells did not show IL-1β immunostaining. Interleukin-1β immunostaining of epidermal cells can discriminate antemortem-postmortem hangings, but vascular structure cells and subepidermal cells cannot discriminate vital hangings.

A cell transportation solution that preserves live circulating tumor cells in patient blood samples.

PubMed

Stefansson, Steingrimur; Adams, Daniel L; Ershler, William B; Le, Huyen; Ho, David H

2016-05-06

Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90% viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs after days of storage. Therefore, we suggest an effective and economical transportation of cancer patient blood samples containing live CTCs can be achieved.

[Validation of Differential Extraction Kit in forensic sexual assault cases].

PubMed

Wu, Dan; Cao, Yu; Xu, Yan; He, Bai-Fang; Bi, Gang; Zhou, Huai-Gu

2009-12-01

To evaluate the validity of Differential Extraction Kit in isolating spermatozoa and epithelial cell DNA from mixture samples. Selective lysis of spermatid and epithelial cells combined with paramagnetic particle method were applied to extract the DNA from the mock samples under controlled conditions and forensic case samples, and template DNA were analyzed by STR genotype method. This Differential Extraction Kit is efficient to obtain high quality spermatid and epithelial cell DNA from the mixture samples with different proportion of sperm to epithelial cell. The Differential Extraction Kit can be applied in DNA extraction for mixed stain from forensic sexual assault samples.

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis.

PubMed

Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Sharma, N S

2015-01-01

The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type. A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV. Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type. It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2.

Dendritic Cells in Kidney Transplant Biopsy Samples Are Associated with T Cell Infiltration and Poor Allograft Survival

PubMed Central

De Serres, Sacha A.; Safa, Kassem; Bijol, Vanesa; Ueno, Takuya; Onozato, Maristela L.; Iafrate, A. John; Herter, Jan M.; Lichtman, Andrew H.; Mayadas, Tanya N.; Guleria, Indira; Rennke, Helmut G.; Najafian, Nader; Chandraker, Anil

2015-01-01

Progress in long-term renal allograft survival continues to lag behind the progress in short-term transplant outcomes. Dendritic cells are the most efficient antigen-presenting cells, but surprisingly little attention has been paid to their presence in transplanted kidneys. We used dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin as a marker of dendritic cells in 105 allograft biopsy samples from 105 kidney transplant recipients. High dendritic cell density was associated with poor allograft survival independent of clinical variables. Moreover, high dendritic cell density correlated with greater T cell proliferation and poor outcomes in patients with high total inflammation scores, including inflammation in areas of tubular atrophy. We then explored the association between dendritic cells and histologic variables associated with poor prognosis. Multivariate analysis revealed an independent association between the densities of dendritic cells and T cells. In biopsy samples with high dendritic cell density, electron microscopy showed direct physical contact between infiltrating lymphocytes and cells that have the ultrastructural morphologic characteristics of dendritic cells. The origin of graft dendritic cells was sought in nine sex-mismatched recipients using XY fluorescence in situ hybridization. Whereas donor dendritic cells predominated initially, the majority of dendritic cells in late allograft biopsy samples were of recipient origin. Our data highlight the prognostic value of dendritic cell density in allograft biopsy samples, suggest a new role for these cells in shaping graft inflammation, and provide a rationale for targeting dendritic cell recruitment to promote long-term allograft survival. PMID:25855773

Multiplexed droplet single-cell RNA-sequencing using natural genetic variation.

PubMed

Kang, Hyun Min; Subramaniam, Meena; Targ, Sasha; Nguyen, Michelle; Maliskova, Lenka; McCarthy, Elizabeth; Wan, Eunice; Wong, Simon; Byrnes, Lauren; Lanata, Cristina M; Gate, Rachel E; Mostafavi, Sara; Marson, Alexander; Zaitlen, Noah; Criswell, Lindsey A; Ye, Chun Jimmie

2018-01-01

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples, demuxlet correctly recovers the sample identity of >99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples.

In vitro biological evaluation of beta-TCP/HDPE--A novel orthopedic composite: a survey using human osteoblast and fibroblast bone cells.

PubMed

Homaeigohar, S Sh; Shokrgozar, M A; Khavandi, A; Sadi, A Yari

2008-02-01

Beta-tricalcium phosphate reinforced high density polyethylene (beta-TCP/HDPE) was prepared to simulate bone composition and to study its capacity to act as bone tissue. This material was produced by replacing the mineral component and collagen soft tissue of the bone with beta-TCP and HDPE, respectively. The biocompatibility of the composite samples with different volume fractions of TCP (20, 30 and 40 vol %) was examined in vitro using two osteoblast cell lines G-292 and Saos-2, and also a type of fibroblast cell isolated from bone tissue, namely human bone fibroblast (HBF) by proliferation, and cell adhesion assays. Cell-material interaction with the surface of the composite samples was examined by scanning electron microscopy (SEM). The effect of beta-TCP/HDPE on the behavior of osteoblast and fibroblast cells was compared with those of composite and negative control samples; polyethylene (PE) and tissue culture polystyrene (TPS), respectively. In general, the results showed that the composite samples containing beta-TCP as reinforcement supported a higher rate of proliferation by various bone cells after 3, 7, and 14 days of incubation compared to the composite control sample. Furthermore, more osteoblast cells were attached to the surface of the composite samples when compared to the composite control samples after the above incubation periods (p < 0.05), while in the case of HBF an equal or even higher number of cells adhered to PE was observed. The number of adhered osteoblast cells was almost equal and in some days even higher than the number of adhered cells on negative control sample, while in the case of fibroblast this difference was significantly higher than TPS (p < 0.05). Adhered cells presented a normal morphology by SEM and many of the cells were observed to be undergoing cell division. These findings indicate that beta-TCP/HDPE composites are biocompatible, nontoxic, and act to stimulate proliferation and adhesion of the cells, whether osteoblast or fibroblast. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.

Application of porous metal enrichment probe sampling to single cell analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).

PubMed

Fu, Qiang; Tang, Jun; Cui, Meng; Xing, Junpeng; Liu, Zhiqiang; Liu, Shuying

2016-01-01

There is an increasing need for analyzing metabolism in a single cell, which is important to understand the nature of cellular heterogeneity, disease, growth and specialization, etc. However, single cell analysis is often challenging for the traces of samples. In the present study, porous metal enrichment probe sampling combined with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) has been applied for in situ analysis of live onion epidemic cell. Porous probe, treated by corroding copper wire with HCl, was directly inserted into a single cell to get cell solution. A self-made linear actuator was enough to control the penetration of probe into the target cell accurately. Then samples on the tip of probe were eluted and detected by a commercial MALDI-TOF-MS directly. The formation of porous microstructure on the probe surface increased the adsorptive capacity of cell solution. The sensitivity of porous probe sampling was 6 times higher than uncorroded probes generally. This method provides a sensitive and convenient way for the sampling and detection of single cell solution. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Hexavalent chromium monitor

DOEpatents

Tao, Shiquan; Winstead, Christopher B.

2005-04-12

A monitor is provided for use in measuring the concentration of hexavalent chromium in a liquid, such as water. The monitor includes a sample cell, a light source, and a photodetector. The sample cell is in the form of a liquid-core waveguide, the sample cell defining an interior core and acting as a receiver for the liquid to be analyzed, the interior surface of the sample cell having a refractive index of less than 1.33. The light source is in communication with a first end of the sample cell for emitting radiation having a wavelength of about and between 350 to 390 nm into the interior core of the waveguide. The photodetector is in communication with a second end of the waveguide for measuring the absorption of the radiation emitted by the light source by the liquid in the sample cell. The monitor may also include a processor electronically coupled to the photodetector for receipt of an absorption signal to determine the concentration of hexavalent chromium in the liquid.

Rapid and simple immunophenotypic characterization of lymphocytes using a new test.

PubMed

Bellido, M; Rubiol, E; Ubeda, J; Estivill, C; López, O; Manteiga, R; Nomdedéu, J F

1998-08-01

In this paper, we report our experience of lymphocyte phenotyping of a series of 108 consecutive samples using a simple flow cytometry test (Lymphogram). The kit consists of a combination of 5 different markers conjugated with three fluorochromes (CD8-FITC, CD19-FITC, CD56-PE, CD3-PE, CD4-PECy5) in the same tube. This allows identification of different T-cells, NK subpopulations and B lymphocytes. The samples were divided into three groups: samples with absolute lymphocytosis (> 5 x 10(9)/L) (n = 50), samples with relative lymphocytosis (> 50%) (n = 24) and other categories for which a lymphocyte immunophenotype was required (T-cell lymphoma and estimation of blood involvement in chronic lymphoproliferative disorders (CLPD) (n = 34). When CD19+ cells exceeded the normal range or there was a suspicion of CLPD without B-cell lymphopenia, clonality was investigated by means of light chain restriction analysis. In the first group, 29 samples were abnormal (10 CLPD, 3 polyclonal B-cell lymphocytosis, 13 inversions of the CD4/CD8 ratio and 3 cases with CD4 lymphocytosis) and 21 samples were regarded as normal. In the second group 7 samples showed abnormalities (2 CLPD, 3 inverted CD4/CD8 ratios and 2 with a relative increase in CD4 cells). In one sample from the third group B-cell clonality without lymphocytosis was detected whereas in 18 samples a polyclonal pattern was observed. The presence of B-cell lymphopenia precluded further clonality study in 13 samples. Lymphogram associated with clonality analysis is a rapid, easy and cheap method of assessing lymphocyte phenotypes in the majority of clinically relevant situations.

Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.

PubMed

Mei, Henrik E; Leipold, Michael D; Schulz, Axel Ronald; Chester, Cariad; Maecker, Holden T

2015-02-15

Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well. Copyright © 2015 by The American Association of Immunologists, Inc.

Fluorescence-Activated Cell Sorting of Live Versus Dead Bacterial Cells and Spores

NASA Technical Reports Server (NTRS)

Bernardini, James N.; LaDuc, Myron T.; Diamond, Rochelle; Verceles, Josh

2012-01-01

This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

Proteomic analysis of single mammalian cells enabled by microfluidic nanodroplet sample preparation and ultrasensitive nanoLC-MS.

PubMed

Zhu, Ying; Clair, Geremy; Chrisler, William; Shen, Yufeng; Zhao, Rui; Shukla, Anil; Moore, Ronald; Misra, Ravi; Pryhuber, Gloria; Smith, Richard; Ansong, Charles; Kelly, Ryan T

2018-05-24

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analysed by ultrasensitive nanoLC-MS. An average of ~670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Glioblastoma stem cell differentiation into endothelial cells evidenced through live-cell imaging.

PubMed

Mei, Xin; Chen, Yin-Sheng; Chen, Fu-Rong; Xi, Shao-Yan; Chen, Zhong-Ping

2017-08-01

Glioblastoma cell-initiated vascularization is an alternative angiogenesis called vasculogenic mimicry. However, current knowledge on the mechanism of de novo vessel formation from glioblastoma stem cells (GSCs) is limited. Sixty-four glioblastoma samples from patients and 10 fluorescent glioma xenograft samples were examined by immunofluorescence staining for endothelial marker (CD34 and CD31) and glial cell marker (glial fibrillary acidic protein [GFAP]) expression. GSCs were then isolated from human glioblastoma tissue and CD133+/Sox2+ red fluorescent protein-containing (RFP)-GSC-1 cells were established. The ability of these cells to form vascular structures was examined by live-cell imaging of 3D cultures. CD34-GFAP or CD31-GFAP coexpressing glioblastoma-derived endothelial cells (GDEC) were found in 30 of 64 (46.9%) of clinical glioblastoma samples. In those 30 samples, GDEC were found to form vessel structures in 21 (70%) samples. Among 21 samples with GDEC vessels, the CD34+ GDEC vessels and CD31+ GDEC vessels accounted for about 14.16% and 18.08% of total vessels, respectively. In the xenograft samples, CD34+ GDEC were found in 7 out of 10 mice, and 4 out of 7 mice had CD34+ GDEC vessels. CD31+ GDEC were also found in 7 mice, and 4 mice had CD31+ GDEC vessels (10 mice in total). Through live-cell imaging, we observed gradual CD34 expression when cultured with vascular endothelial growth factor in some glioma cells, and a dynamic increase in endothelial marker expression in RFP-GSC-1 in vitro was recorded. Cells expressed CD34 (9.46%) after 6 hours in culture. The results demonstrated that GSCs may differentiate into endothelial cells and promote angiogenesis in glioblastomas. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

21 CFR 660.36 - Samples and protocols.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.36 Samples... a cell panel intended for identification of unexpected antibodies. The sample shall be packaged as... distribution of each lot of Reagent Red Blood Cells for detection or identification of unexpected antibodies...

Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass

NASA Technical Reports Server (NTRS)

Benardini, James N.; LaDuc, Myron T.; Diamond, Rochelle

2012-01-01

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.

Expression of microphthalmia transcription factor, S100 protein, and HMB-45 in malignant melanoma and pigmented nevi.

PubMed

Xia, Jianxin; Wang, Yanlong; Li, Fuqiu; Wang, Jinfeng; Mu, Yan; Mei, Xianglin; Li, Xue; Zhu, Wenjing; Jin, Xianhua; Yu, Kai

2016-09-01

Malignant melanoma (MM) is a type of malignant tumor, which originates from neural crest melanocytes. MM progresses rapidly and results in a high mortality rate. The present study aims to investigate the expression of microphthalmia transcription factor (MITF), the S100 protein, and HMB-45 in MM and pigmented nevi. A total of 32 MM samples (including three skin metastasis, three lymph node metastasis and two spindle cell MM samples), two Spitz nevus samples, four pigmented nevus samples and two blue nevus samples were collected. The expression levels of S100 protein, HMB-45, and MITF were observed via immunostaining. The S100 protein exhibited high positive rates in MM and pigment disorders (96.7 and 100%, respectively), but with low specificity. The S100 protein was also expressed in fibroblasts, myoepithelial cells, histocytes and Langerhans cells in normal skin samples. HMB-45 had high specificity. Its positive expression was only confined to MM cells and junctional nevus cells. Furthermore, HMB-45 was not expressed in melanocytes in the normal tissue samples around the tumor or in the benign intradermal nevus cells. MITF exhibited high specificity and high sensitivity. It was expressed in the nuclei of melanocytes, MM cells and nevus cells. It was observed to be strongly expressed in metastatic MM and spindle cell MMs. Thus, MITF may present as a specific immunomarker for the diagnosis and differential diagnosis of MM.

Universal nucleic acids sample preparation method for cells, spores and their mixture

DOEpatents

Bavykin, Sergei [Darien, IL

2011-01-18

The present invention relates to a method for extracting nucleic acids from biological samples. More specifically the invention relates to a universal method for extracting nucleic acids from unidentified biological samples. An advantage of the presently invented method is its ability to effectively and efficiently extract nucleic acids from a variety of different cell types including but not limited to prokaryotic or eukaryotic cells and/or recalcitrant organisms (i.e. spores). Unlike prior art methods which are focused on extracting nucleic acids from vegetative cell or spores, the present invention effectively extracts nucleic acids from spores, multiple cell types or mixtures thereof using a single method. Important that the invented method has demonstrated an ability to extract nucleic acids from spores and vegetative bacterial cells with similar levels effectiveness. The invented method employs a multi-step protocol which erodes the cell structure of the biological sample, isolates, labels, fragments nucleic acids and purifies labeled samples from the excess of dye.

Fluorometric determination of the DNA concentration in municipal drinking water.

PubMed Central

McCoy, W F; Olson, B H

1985-01-01

DNA concentrations in municipal drinking water samples were measured by fluorometry, using Hoechst 33258 fluorochrome. The concentration, extraction, and detection methods used were adapted from existing techniques. The method is reproducible, fast, accurate, and simple. The amounts of DNA per cell for five different bacterial isolates obtained from drinking water samples were determined by measuring DNA concentration and total cell concentration (acridine orange epifluorescence direct cell counting) in stationary pure cultures. The relationship between DNA concentration and epifluorescence total direct cell concentration in 11 different drinking water samples was linear and positive; the amounts of DNA per cell in these samples did not differ significantly from the amounts in pure culture isolates. We found significant linear correlations between DNA concentration and colony-forming unit concentration, as well as between epifluorescence direct cell counts and colony-forming unit concentration. DNA concentration measurements of municipal drinking water samples appear to monitor changes in bacteriological quality at least as well as total heterotrophic plate counting and epifluorescence direct cell counting. PMID:3890737

The counting of native blood cells by digital microscopy

NASA Astrophysics Data System (ADS)

Torbin, S. O.; Doubrovski, V. A.; Zabenkov, I. V.; Tsareva, O. E.

2017-03-01

An algorithm for photographic images processing of blood samples in its native state was developed to determine the concentration of erythrocytes, leukocytes and platelets without individual separate preparation of cells' samples. Special "photo templates" were suggested to use in order to identify red blood cells. The effect of "highlighting" of leukocytes, which was found by authors, was used to increase the accuracy of this type of cells counting. Finally to raise the resolution of platelets from leukocytes the areas of their photo images were used, but not their sizes. It is shown that the accuracy of cells counting for native blood samples may be comparable with the accuracy of similar studies for smears. At the same time the proposed native blood analysis simplifies greatly the procedure of sample preparation in comparison to smear, permits to move from the detection of blood cells ratio to the determination of their concentrations in the sample.

The effect of CO2 laser beam welded AISI 316L austenitic stainless steel on the viability of fibroblast cells, in vitro.

PubMed

Köse, Ceyhun; Kaçar, Ramazan; Zorba, Aslı Pınar; Bağırova, Melahat; Allahverdiyev, Adil M

2016-03-01

It has been determined by the literature research that there is no clinical study on the in vivo and in vitro interaction of the cells with the laser beam welded joints of AISI 316L biomaterial. It is used as a prosthesis and implant material and that has adequate mechanical properties and corrosion resistance characteristics. Therefore, the interaction of the CO2 laser beam welded samples and samples of the base metal of AISI 316L austenitic stainless steel with L929 fibroblast cells as an element of connective tissue under in vitro conditions has been studied. To study the effect of the base metal and the laser welded test specimens on the viability of the fibroblast cells that act as an element of connective tissues in the body, they were kept in DMEMF-12 medium for 7, 14, 28 days and 18 months. The viability study was experimentally studied using the MTT method for 7, 14, 28 days. In addition, the direct interaction of the fibroblast cells seeded on 6 different plates with the samples was examined with an inverted microscope. The MTT cell viability experiment was repeated on the cells that were in contact with the samples. The statistical relationship was analyzed using a Tukey test for the variance with the GraphPad statistics software. The data regarding metallic ion release were identified with the ICP-MS method after the laser welded and main material samples were kept in cell culture medium for 18 months. The cell viability of the laser welded sample has been detected to be higher than that of the base metal and the control based on 7th day data. However, the laser welded sample's viability of the fibroblast cells has diminished by time during the test period of 14 and 28 days and base metal shows better viability when compared to the laser welded samples. On the other hand, the base metal and the laser welded sample show better cell viability effect when compared to the control group. According to the ICP-MS results of the main material and laser welded samples which were kept in the cell culture medium for 18 months, it was determined that the Fe, Ni and Cr ion concentration released to the cell culture medium from the laser welded test sample was less than that of the main material. Copyright © 2015 Elsevier B.V. All rights reserved.

Design of the sample cell in near-field surface-enhanced Raman scattering by finite difference time domain method

NASA Astrophysics Data System (ADS)

Li, Yaqin; Jian, Guoshu; Wu, Shifa

2006-11-01

The rational design of the sample cell may improve the sensitivity of surface-enhanced Raman scattering (SERS) detection in a high degree. Finite difference time domain (FDTD) simulations of the configuration of Ag film-Ag particles illuminated by plane wave and evanescent wave are performed to provide physical insight for design of the sample cell. Numerical solutions indicate that the sample cell can provide more "hot spots' and the massive field intensity enhancement occurs in these "hot spots'. More information on the nanometer character of the sample can be got because of gradient-field Raman (GFR) of evanescent wave.

The effect of partially stabilized zirconia on the biological properties of HA/HDPE composites in vitro.

PubMed

Sadi, A Yari; Shokrgozar, M A; Homaeigohar, S Sh; Hosseinalipour, M; Khavandi, A; Javadpour, J

2006-05-01

The effect of partially stabilized zirconia (PSZ) on the biological properties of the hyroxyapatite - high density polyethylene (HA/HDPE) composites was studied by investigating the simultaneous effect of hydroxyapatite and PSZ volume fractions on the in vitro response of human osteoblast cells. The biocompatibility of composite samples with different volume fraction of HA and PSZ powders was assessed by proliferation, alkaline phosphatase (ALP) and cell attachment assays on the osteoblast cell line (G-292) in different time periods. The effect of composites on the behavior of G-292 cells was compared with those of HDPE and TPS (Tissue Culture Poly Styrene as negative control) samples. Results showed a higher proliferation rate of G-292 cells in the presence of composite samples as compared to the HDPE sample after 7 and 14 days of incubation period. ALP production rate in all composite samples was higher than HDPE and TPS samples. The number of adhered cells on the composite samples was higher than the number adhered on the HDPE and TPS samples after the above mentioned incubation periods. These findings indicates that the addition of PSZ does not have any adverse affect on the biocompatibility of HA/HDPE composites. In fact in some experiments PSZ added HA/HDPE composites performed better in proliferation, differentiation and attachment of osteoblastic cells.

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis

PubMed Central

Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P. N.; Sharma, N. S.

2015-01-01

Aim: The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type. Materials and Methods: A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV. Results: Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type. Conclusion: It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2. PMID:27046996

Microfluidics apparatus and methods for use thereof

DOEpatents

Peeters, John P.; Wiggins, Thomas; Ghosh, Madhushree; Bottomley, Lawrence A.; Seminara, Salvatore; Hu, Zhiyu; Seeley, Timothy; Kossek, Sebastian

2005-08-09

A microfluidics device includes a plurality of interaction cells and fluid control means including i) means for providing to the interaction cells a preparation fluid, and ii) means for providing to the interaction cells a sample fluid, wherein each interaction cell receives a different sample fluid. A plurality of microcantilevers may be disposed in each of the interaction cells, wherein each of the plurality of microcantilevers configured to deflect in response to an interaction involving a component of the sample fluid.

Massively parallel nanowell-based single-cell gene expression profiling.

PubMed

Goldstein, Leonard D; Chen, Ying-Jiun Jasmine; Dunne, Jude; Mir, Alain; Hubschle, Hermann; Guillory, Joseph; Yuan, Wenlin; Zhang, Jingli; Stinson, Jeremy; Jaiswal, Bijay; Pahuja, Kanika Bajaj; Mann, Ishminder; Schaal, Thomas; Chan, Leo; Anandakrishnan, Sangeetha; Lin, Chun-Wah; Espinoza, Patricio; Husain, Syed; Shapiro, Harris; Swaminathan, Karthikeyan; Wei, Sherry; Srinivasan, Maithreyan; Seshagiri, Somasekar; Modrusan, Zora

2017-07-07

Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.

Susceptibility-matched plugs for microcoil NMR probes

NASA Astrophysics Data System (ADS)

Kc, Ravi; Gowda, Yashas N.; Djukovic, Danijel; Henry, Ian D.; Park, Gregory H. J.; Raftery, Daniel

2010-07-01

For mass-limited samples, the residual sample volume outside the detection coil is an important concern, as is good base line resolution. Here, we present the construction and evaluation of magnetic susceptibility-matched plugs for microcoil NMR sample cells which address these issues. Mixed-epoxy glue and ultem tube plugs that have susceptibility values close to those of perfluorocarbon FC-43 (fluorinert) and copper were used in small volume (0.5-2 μL) and larger volume (15-20 μL) thin glass capillary sample cells. Using these plugs, the sample volume efficiency (i.e. ratio of active volume to total sample volume in the microcoil NMR cell) was improved by 6-12-fold without sensitivity and resolution trade-offs. Comparison with laser etched or heat etched microcoil sample cells is provided. The approaches described are potentially useful in metabolomics for biomarkers detection in mass limited biological samples.

Susceptibility-matched plugs for microcoil NMR probes.

PubMed

Kc, Ravi; Gowda, Yashas N; Djukovic, Danijel; Henry, Ian D; Park, Gregory H J; Raftery, Daniel

2010-07-01

For mass-limited samples, the residual sample volume outside the detection coil is an important concern, as is good base line resolution. Here, we present the construction and evaluation of magnetic susceptibility-matched plugs for microcoil NMR sample cells which address these issues. Mixed-epoxy glue and ultem tube plugs that have susceptibility values close to those of perfluorocarbon FC-43 (fluorinert) and copper were used in small volume (0.5-2 microL) and larger volume (15-20 microL) thin glass capillary sample cells. Using these plugs, the sample volume efficiency (i.e. ratio of active volume to total sample volume in the microcoil NMR cell) was improved by 6-12-fold without sensitivity and resolution trade-offs. Comparison with laser etched or heat etched microcoil sample cells is provided. The approaches described are potentially useful in metabolomics for biomarkers detection in mass limited biological samples. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Susceptibility-matched plugs for microcoil NMR probes

PubMed Central

Kc, Ravi; Gowda, Yashas N.; Djukovic, Danijel; Henry, Ian D; Park, Gregory H J; Raftery, Daniel

2010-01-01

For mass limited samples, the residual sample volume outside the detection coil is an important concern, as is good base line resolution. Here, we present the construction and evaluation of magnetic susceptibility-matched plugs for microcoil NMR sample cells which address these issues. Mixed-epoxy glue and ultem tube plugs that have susceptibility values close to those of perfluorocarbon FC-43 (fluorinert) and copper were used in small volume (0.5 to 2 μL) and larger volume (15 to 20 μL) thin glass capillary sample cells. Using these plugs, the sample volume efficiency (i.e. ratio of active volume to total sample volume in the microcoil NMR cell) was improved by 6 to 12 fold without sensitivity and resolution trade-offs. Comparison with laser etched or heat etched microcoil sample cells is provided. The approaches described are potentially useful in metabolomics for biomarkers detection in mass limited biological samples. PMID:20510638

Isolation of influenza virus in human lung embryonated fibroblast cells (MRC-5) from clinical samples.

PubMed Central

de Oña, M; Melón, S; de la Iglesia, P; Hidalgo, F; Verdugo, A F

1995-01-01

Ninety-four pharyngeal swab samples corresponding to 94 patients with suspected influenza virus infection were inoculated in Madin-Darby canine kidney (MDCK) cells, the conventional cell system for the isolation of influenza virus, and in fibroblastic human embryo lung (MRC-5) cells, a cell system less commonly used for this purpose but one frequently used in clinical virology laboratories. Both cell preparations were treated with trypsin. Influenza virus was recovered from 15% of the samples inoculated in MDCK cells and from 18% of those inoculated in MRC-5 cells. The use of MRC-5 cells can simplify the search for respiratory viruses and would assist in the rapid detection of influenza virus during new epidemics. PMID:7665680

Profiling pleural effusion cells by a diffraction imaging method

NASA Astrophysics Data System (ADS)

Al-Qaysi, Safaa; Hong, Heng; Wen, Yuhua; Lu, Jun Q.; Feng, Yuanming; Hu, Xin-Hua

2018-02-01

Assay of cells in pleural effusion (PE) is an important means of disease diagnosis. Conventional cytology of effusion samples, however, has low sensitivity and depends heavily on the expertise of cytopathologists. We applied a polarization diffraction imaging flow cytometry method on effusion cells to investigate their features. Diffraction imaging of the PE cell samples has been performed on 6000 to 12000 cells for each effusion cell sample of three patients. After prescreening to remove images by cellular debris and aggregated non-cellular particles, the image textures were extracted with a gray level co-occurrence matrix (GLCM) algorithm. The distribution of the imaged cells in the GLCM parameters space was analyzed by a Gaussian Mixture Model (GMM) to determine the number of clusters among the effusion cells. These results yield insight on textural features of diffraction images and related cellular morphology in effusion samples and can be used toward the development of a label-free method for effusion cells assay.

Tissue response to peritoneal implants

NASA Technical Reports Server (NTRS)

Picha, G. J.

1980-01-01

Peritoneal implants were fabricated from poly 2-OH, ethyl methacrylate (HEMA), polyetherurethane (polytetramethylene glycol 1000 MW, 1,4 methylene disocynate, and ethyl diamine), and untreated and sputter treated polytetrafluoroethylene (PTFE). The sputter treated PTFE implants were produced by an 8 cm diameter argon ion source. The treated samples consisted of ion beam sputter polished samples, sputter etched samples (to produce a microscopic surface cone texture) and surface pitted samples (produced by ion beam sputtering to result in 50 microns wide by 100 microns deep square pits). These materials were implanted in rats for periods ranging from 30 minutes to 14 days. The results were evaluated with regard to cell type and attachment kinetics onto the different materials. Scanning electron microscopy and histological sections were also evaluated. In general the smooth hydrophobic surfaces attracted less cells than the ion etched PTFE or the HEMA samples. The ion etching was observed to enhance cell attachment, multinucleated giant cell (MNGC) formation, cell to cell contact, and fibrous capsule formation. The cell responsed in the case of ion etched PTFE to an altered surface morphology. However, equally interesting was the similar attachment kinetics of HEMA verses the ion etched PTFE. However, HEMA resulted in a markedly different response with no MNGC's formation, minimal to no capsule formation, and sample coverage by a uniform cell layer.

Standardized cell samples for midIR technology development

NASA Astrophysics Data System (ADS)

Kastl, Lena; Rommel, Christina E.; Kemper, Björn; Schnekenburger, Jürgen

2015-03-01

The application of midIR spectroscopy towards human cell and tissue samples is impaired by the need for technical solutions and lacking sample standards for technology development. We here present the standardization of stable test samples for the continuous development and testing of novel optical system components. We have selected cell lines representing the major cellular skin constituents keratinocytes and fibroblasts (NIH-3T3, HaCaT). In addition, two skin cancer cell types (A-375 and SK-MEL-28 cells) were analyzed. Cells were seeded on CaF2 substrates and measured dried and under aqueous medium as well as fixated and unfixated. Several independent cell preparations were analyzed with an FTIR spectrometer in the wave number range from 1000 - 4000 cm-1. The obtained data demonstrate that fixed and dehydrated cells on CaF2 can be stored in pure ethanol for several weeks without significant losses in quality of the spectral properties. The established protocol of cell seeding on CaF2 substrates, chemical fixation, dehydration, storage under ethanol and air-drying is suitable for the production of reliable midIR standards. The retrieved spectra demonstrate that fixed cells on CaF2 can be prepared reproducibly; with stable midIR spectral properties over several weeks and properties mimicking reliable unfixed cells. Moreover, the fixated samples on CaF2 show clear differences in the cell type specific spectra that can be identified by principle component analysis. In summary, the standardized cell culture samples on CaF2 substrates are suitable for the development of a midIR device and the optimization of the specific midIR spectra.

Barcoding of live human PBMC for multiplexed mass cytometry*

PubMed Central

Mei, Henrik E.; Leipold, Michael D.; Schulz, Axel Ronald; Chester, Cariad; Maecker, Holden T.

2014-01-01

Mass cytometry is developing as a means of multiparametric single cell analysis. Here, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a CyTOF® instrument. Using six different anti-CD45 antibody (Ab) conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and reduces wet work and antibody consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45-barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and should be applicable to fluorescence flow cytometry as well. PMID:25609839

Comparison of three sampling instruments, Cytobrush, Curette and OralCDx, for liquid-based cytology of the oral mucosa.

PubMed

Reboiras-López, M D; Pérez-Sayáns, M; Somoza-Martín, J M; Antúnez-López, J R; Gándara-Vila, P; Gayoso-Diz, P; Gándara-Rey, J M; García-García, A

2012-01-01

Exfoliative cytology of the oral cavity is a simple and noninvasive technique that permits the study of epithelial cells. Liquid-based cytology is an auxiliary diagnostic tool for improving the specificity and sensitivity of conventional cytology. The objective of our study was to compare the quality of normal oral mucosa cytology samples obtained using three different instruments, Cytobrush®, dermatological curette and Oral CDx® for liquid-based cytology. One hundred four cytological samples of oral cavity were analyzed. Samples were obtained from healthy volunteer subjects using all three instruments. The clinical and demographic variables were age, sex and smoking habits. We analyzed cellularity, quality of the preparation and types of cells in the samples. All preparations showed appropriate preparation quality. In all smears analyzed, cells were distributed uniformly and showed no mucus, bleeding, inflammatory exudate or artifacts. We found no correlation between the average number of cells and the type of instrument. The samples generally consisted of two types of cells: superficial and intermediate. No differences were found among the cytological preparations of these three instruments. We did not observe basal cells in any of the samples analyzed.

Evaluation of adsorption effects on measurements of ammonia, acetic acid, and methanol

NASA Astrophysics Data System (ADS)

Yokelson, R. J.; Christian, T. J.; Bertschi, I. T.; Hao, W. M.

2003-10-01

We examined how adsorption and desorption of gases from inlets and a cell could affect the accuracy of closed-cell FTIR measurements of carbon dioxide (CO2), carbon monoxide (CO), methane (CH4), nitric oxide (NO), nitrogen dioxide (NO2), methanol (CH3OH), acetic acid (CH3COOH), and ammonia (NH3). When standards were delivered to the cell through a stainless steel inlet, temporarily reduced transmission was observed for CH3OH and NH3. However, a halocarbon wax coated inlet (normally used on the system) had excellent transmission (comparable to room temperature Teflon) for both CH3OH and NH3, even at temperatures as low as 5°C. Thus the wax is valuable for coating sampling system components that cannot be fashioned from Teflon. The instrument had a delayed response (˜10-40 s) for NH3 only, which was attributed to passivation of the Pyrex multipass cell. To determine sampling artifacts that could arise from the complex sample matrix presented by smoke, the closed-cell FTIR system was intercompared with an open-path FTIR system (which is immune to sampling artifacts) in well-mixed smoke. A similar cell passivation delay for NH3 was the only artifact found in this test. Overall, the results suggest that ˜10 s is sufficient to detect >80% of an NH3/CO ratio sampled by our fast-flow, closed-cell system. Longer sampling times or consecutive samples return better results. In field campaigns the closed-cell system sampling times were normally 10 to >100 s so NH3 was probably underestimated by 5-15%.

Design and evaluation of a new Peltier-cooled laser ablation cell with on-sample temperature control.

PubMed

Konz, Ioana; Fernández, Beatriz; Fernández, M Luisa; Pereiro, Rosario; Sanz-Medel, Alfredo

2014-01-27

A new custom-built Peltier-cooled laser ablation cell is described. The proposed cryogenic cell combines a small internal volume (20 cm(3)) with a unique and reliable on-sample temperature control. The use of a flexible temperature sensor, directly located on the sample surface, ensures a rigorous sample temperature control throughout the entire analysis time and allows instant response to any possible fluctuation. In this way sample integrity and, therefore, reproducibility can be guaranteed during the ablation. The refrigeration of the proposed cryogenic cell combines an internal refrigeration system, controlled by a sensitive thermocouple, with an external refrigeration system. Cooling of the sample is directly carried out by 8 small (1 cm×1 cm) Peltier elements placed in a circular arrangement in the base of the cell. These Peltier elements are located below a copper plate where the sample is placed. Due to the small size of the cooling electronics and their circular allocation it was possible to maintain a peephole under the sample for illumination allowing a much better visualization of the sample, a factor especially important when working with structurally complex tissue sections. The analytical performance of the cryogenic cell was studied using a glass reference material (SRM NIST 612) at room temperature and at -20°C. The proposed cell design shows a reasonable signal washout (signal decay within less than 10 s to background level), high sensitivity and good signal stability (in the range 6.6-11.7%). Furthermore, high precision (0.4-2.6%) and accuracy (0.3-3.9%) in the isotope ratio measurements were also observed operating the cell both at room temperature and at -20°C. Finally, experimental results obtained for the cell application to qualitative elemental imaging of structurally complex tissue samples (e.g. eye sections from a native frozen porcine eye and fresh flower leaves) demonstrate that working in cryogenic conditions is critical in such type of direct sample analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for Foot-and-mouth disease virus isolation.

PubMed

Fukai, Katsuhiko; Morioka, Kazuki; Yamada, Manabu; Nishi, Tatsuya; Yoshida, Kazuo; Kitano, Rie; Yamazoe, Reiko; Kanno, Toru

2015-07-01

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-α(v)β(6) have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-α(v)β(6) cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-α(v)β(6) cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same. © 2015 The Author(s).

Magnetic bead-based separation of sperm from buccal epithelial cells using a monoclonal antibody against MOSPD3.

PubMed

Li, Xue-Bo; Wang, Qing-Shan; Feng, Yu; Ning, Shu-Hua; Miao, Yuan-Ying; Wang, Ye-Quan; Li, Hong-Wei

2014-11-01

Forensic DNA analysis of sexual assault evidence requires unambiguous differentiation of DNA profiles in mixed samples. To investigate the feasibility of magnetic bead-based separation of sperm from cell mixtures using a monoclonal antibody against MOSPD3 (motile sperm domain-containing protein 3), 30 cell samples were prepared by mixing 10(4) female buccal epithelial cells with sperm cells of varying densities (10(3), 10(4), or 10(5) cells/mL). Western blot and immunofluorescence assays showed that MOSPD3 was detectable on the membrane of sperm cells, but not in buccal epithelial cells. After biotinylated MOSPD3 antibody was incubated successively with the prepared cell mixtures and avidin-coated magnetic beads, microscopic observation revealed that each sperm cell was bound by two or more magnetic beads, in the head, neck, mid-piece, or flagellum. A full single-source short tandem repeat profile could be obtained in 80% of mixed samples containing 10(3) sperm cells/mL and in all samples containing ≥10(4) sperm cells/mL. For dried vaginal swab specimens, the rate of successful detection was 100% in both flocked and cotton swabs preserved for 1 day, 87.5% in flocked swabs and 40% in cotton swabs preserved for 3 days, and 40% in flocked swabs and 16.67% in cotton swabs preserved for 10 days. Our findings suggest that immunomagnetic bead-based separation is potentially a promising alternative to conventional methods for isolating sperm cells from mixed forensic samples.

MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples.

PubMed

Sivaganesan, Mano; Siefring, Shawn; Varma, Manju; Haugland, Richard A

2011-12-01

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for uncertainty, the high degree of precision attainable by the MPN approach and the need to account for the differences in target sequence recoveries from different calibrator sample cell sources when they are used in the comparative Ct method. Published by Elsevier B.V.

Clinical evaluation of a novel microfluidic device for epitope-independent enrichment of circulating tumour cells in patients with small cell lung cancer.

PubMed

Chudziak, Jakub; Burt, Deborah J; Mohan, Sumitra; Rothwell, Dominic G; Mesquita, Bárbara; Antonello, Jenny; Dalby, Suzanne; Ayub, Mahmood; Priest, Lynsey; Carter, Louise; Krebs, Matthew G; Blackhall, Fiona; Dive, Caroline; Brady, Ged

2016-01-21

Circulating tumour cells (CTCs) have potential utility as minimally-invasive biomarkers to aid cancer treatment decision making. However, many current CTC technologies enrich CTCs using specific surface epitopes that do not necessarily reflect CTC heterogeneity. Here we evaluated the epitope-independent Parsortix system which enriches CTCs based on size and rigidity using both healthy normal volunteer blood samples spiked with tumour cells and blood samples from patients with small cell lung cancer (SCLC). Blood samples were maintained unfractionated at room temperature for up to 4 days followed by plasma removal for circulating free DNA (cfDNA) isolation and direct application of the remaining cell component to the Parsortix system. For tumour cells expressing the EpCAM cell surface marker the numbers of spiked cells retained using the Parsortix system and by EpCAM-positive selection using CellSearch® were not significantly different, whereas only the Parsortix system showed strong enrichment of cells with undetectable EpCAM expression. In a pilot clinical study we banked both enriched CTCs as well as plasma from SCLC patient blood samples. Upon retrieval of the banked Parsortix cellular samples we could detect cytokeratin positive CTCs in all 12 SCLC patients tested. Interestingly, processing parallel samples from the same patients by EpCAM enrichment using CellSearch® revealed only 83% (10/12) with cytokeratin positive CTCs indicating the Parsortix system is enriching for EpCAM negative SCLC CTCs. Our combined results indicate the Parsortix system is a valuable tool for combined cfDNA isolation and CTC enrichment that enables CTC analysis to be extended beyond dependence on surface epitopes.

A systematic assessment of goblet cell sampling of the bulbar conjunctiva by impression cytology.

PubMed

Doughty, Michael J

2015-07-01

The purpose of this study was to assess the apparent goblet cell density (GCD) from conjunctival impression cytology (CIC) samples in relation to the number of conjunctival cells collected onto the filters. CIC specimens were collected from the superior-temporal bulbar conjunctiva of 16 pigmented rabbits onto Biopore (Millicell-CM) membranes, fixed with buffered glutaraldehyde and stained with Giemsa. Different numbers of microscope fields of view in each of the specimens were imaged by light microscopy using a 20× magnification objective lens (200× final magnification), and the goblet cells marked and counted. The GCD values/sq. mm were calculated. The same conjunctival region of 3 other rabbits was also prepared for transmission electron microscopy (TEM) by fixation, in situ, with the same buffered glutaraldehyde. Mean values for GCD estimates were found to vary from 399 to 1576 cells/sq. mm, depending on the image sampling and analysis strategy chosen, with the lowest inter-sample variance of around 10% being found if a maximum goblet cell count was taken on substantially multilayered regions of the CIC specimens. Counts of the number of goblet cells per 1000 visible conjunctival epithelial cells yielded a value of close to 90 (range 36-151), with modest inter-sample variability of around 30%. A three or ten 200× microscope field and random sampling strategy yielded mean GCD values between 542 and 670 cells/sq. mm, but with very high intra- and inter-sample variance of at least 60% and sometimes higher than 100%. TEM confirmed the multilayered organization of the conjunctiva and the deeper lying goblet cells. The general use of a goblet cell count as an objective marker for conjunctival normality or health is likely to be highly variable unless a more specific strategy is adopted. Beyond providing details of exactly the counting strategy used, it would be very useful to provide full details of the actual microscope field size used as well as information on the intra-sample variability in goblet cell counts. Copyright © 2015 Elsevier Ltd. All rights reserved.

Extraction of nucleic acids from yeast cells and plant tissues using ethanol as medium for sample preservation and cell disruption.

PubMed

Linke, Bettina; Schröder, Kersten; Arter, Juliane; Gasperazzo, Tatiana; Woehlecke, Holger; Ehwald, Rudolf

2010-09-01

Here we report that dehydrated ethanol is an excellent medium for both in situ preservation of nucleic acids and cell disruption of plant and yeast cells. Cell disruption was strongly facilitated by prior dehydration of the ethanol using dehydrated zeolite. Following removal of ethanol, nucleic acids were extracted from the homogenate pellet using denaturing buffers. The method provided DNA and RNA of high yield and integrity. Whereas cell wall disruption was essential for extraction of DNA and large RNA molecules, smaller molecules such as tRNAs could be selectively extracted from undisrupted, ethanol-treated yeast cells. Our results demonstrate the utility of absolute ethanol for sample fixation, cell membrane and cell wall disruption, as well as preservation of nucleic acids during sample storage.

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics.

PubMed

Tung, Yi-Chung; Torisawa, Yu-suke; Futai, Nobuyuki; Takayama, Shuichi

2007-11-01

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.

A general method for bead-enhanced quantitation by flow cytometry

PubMed Central

Montes, Martin; Jaensson, Elin A.; Orozco, Aaron F.; Lewis, Dorothy E.; Corry, David B.

2009-01-01

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry. PMID:17067632

Multiple sample characterization of coals and other substances by controlled-atmosphere programmed temperature oxidation

DOEpatents

LaCount, Robert B.

1993-01-01

A furnace with two hot zones holds multiple analysis tubes. Each tube has a separable sample-packing section positioned in the first hot zone and a catalyst-packing section positioned in the second hot zone. A mass flow controller is connected to an inlet of each sample tube, and gas is supplied to the mass flow controller. Oxygen is supplied through a mass flow controller to each tube to either or both of an inlet of the first tube and an intermediate portion between the tube sections to intermingle with and oxidize the entrained gases evolved from the sample. Oxidation of those gases is completed in the catalyst in each second tube section. A thermocouple within a sample reduces furnace temperature when an exothermic condition is sensed within the sample. Oxidized gases flow from outlets of the tubes to individual gas cells. The cells are sequentially aligned with an infrared detector, which senses the composition and quantities of the gas components. Each elongated cell is tapered inward toward the center from cell windows at the ends. Volume is reduced from a conventional cell, while permitting maximum interaction of gas with the light beam. Reduced volume and angulation of the cell inlets provide rapid purgings of the cell, providing shorter cycles between detections. For coal and other high molecular weight samples, from 50% to 100% oxygen is introduced to the tubes.

Authentication of M14 melanoma cell line proves misidentification of MDA‐MB‐435 breast cancer cell line

PubMed Central

Korch, Christopher; Hall, Erin M.; Dirks, Wilhelm G.; Ewing, Margaret; Faries, Mark; Varella‐Garcia, Marileila; Robinson, Steven; Storts, Douglas; Turner, Jacqueline A.; Wang, Ying; Burnett, Edward C.; Healy, Lyn; Kniss, Douglas; Neve, Richard M.; Nims, Raymond W.; Reid, Yvonne A.; Robinson, William A.

2017-01-01

A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research. PMID:28940260

Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis.

PubMed

Li, Zi-Yi; Huang, Min; Wang, Xiu-Kun; Zhu, Ying; Li, Jin-Song; Wong, Catherine C L; Fang, Qun

2018-04-17

Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic analysis in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (>99%), thus substantially increasing the analytical sensitivity for single cell samples. We applied the present system in the proteomic analysis of 100 ± 10, 50 ± 5, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, respectively. The OAD chip-based system was further applied in single mouse oocyte analysis, with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymatic digestion efficiency over the traditional in-tube system.

Laser dissection sampling modes for direct mass spectral analysis [using a hybrid optical microscopy/laser ablation liquid vortex capture/electrospray ionization system

DOE PAGES

Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

2016-02-01

Here, laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis ofmore » single cells and tissue. Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (4-15 m) even when agglomerated together. Turbid Allium Cepa cells (150 m) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. In conclusion, laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.« less

Laser dissection sampling modes for direct mass spectral analysis [using a hybrid optical microscopy/laser ablation liquid vortex capture/electrospray ionization system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

Here, laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis ofmore » single cells and tissue. Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (4-15 m) even when agglomerated together. Turbid Allium Cepa cells (150 m) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. In conclusion, laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.« less

Cavity ring-down spectroscopy in the liquid phase

NASA Astrophysics Data System (ADS)

Xu, Shucheng; Sha, Guohe; Xie, Jinchun

2002-02-01

A new application for cavity ring-down spectroscopic (CRDS) technique using a pulsed polarized light source has been developed in the absorption measurement of liquids for "colorless" organic compounds using both a single sample cell and double sample cells inserted in an optical cavity at Brewster angle. At present an experimental capability of measuring absorption coefficients as small as 2-5×10-7 cm-1 has been demonstrated by measurement of the absorption baselines. The first spectra for CRDS in the liquid phase, the C-H stretching fifth vibrational overtones of benzene in the pure liquid and hexane solution are obtained. The optical absorption length for liquids in both a single sample cell and double sample cells of 1 cm length is up to 900 cm due to multipass of light within an optical cavity. Compared to the thermal lens and optoacoustic spectroscopic techniques, the sensitivity for CRDS mainly depends on the optical absorption path of the sample (single passing path of the sample times multipass times), is not determined by the laser power and the length of the sample cell. The absolute absorption coefficient and band intensity for the sample are determined directly by the spectroscopy.

The contribution of mammary infections by coagulase-negative staphylococci to the herd bulk milk somatic cell count.

PubMed

Rainard, P; Ducelliez, M; Poutrel, B

1990-01-01

Quarter foremilk samples were taken at 2-3 weekly intervals for several years in an experimental herd comprising about 45 cows. The samples were submitted to bacteriological analysis and somatic cell counting. The most prevalent quarter infections from 1982 to 1988 were by coagulase-negative staphylococci (15-20% of all the quarters sampled). Most of these (75.6%) persisted until drying-off. Dry cow therapy eliminated 86.5% of these infections. Comparison of udder quarters within cows, involving 775 samples from pairs of non-infected quarters and quarters infected by coagulase-negative staphylococci, yielded geometric means of somatic cell counts of 210,000 and 420,000 cells/ml, respectively. The correlation (r = 0.87) between the herd bulk milk somatic cell count (SCC) and its estimation from the quarter milk somatic cell count performed on the same day allowed us to evaluate the contribution of the different categories of quarters, according to their infection status, to the herd bulk milk SCC. Quarters infected by a major pathogen (8.5% of samples) gave rise to 46.6% of the total number of cells, while quarters infected by coagulase-negative staphylococci (17.8% of samples) gave rise to 18.1%. Although coagulase-negative staphylococci represented only a secondary source of somatic cells as compared to major pathogens, they were not a negligible source considering the threshold of 300,000 somatic cells advocated for herd milk of good quality.

Simultaneous immunostaining with anti-S100P and anti-SV40 antibodies revealed the origin of BK virus-infected decoy cells in voided urine samples.

PubMed

Ariyasu, S; Yanai, H; Sato, M; Shinno, Y; Taniguchi, K; Yamadori, I; Miki, Y; Sato, Y; Yoshino, T; Takahashi, K

2015-08-01

Methods for determining the origin of BK virus (BKV)-infected cells (decoy cells) in clinical urine samples have not been established although they could enhance the diagnosis of BKV infection in immunocompromised patients. We performed simultaneous immunostaining with anti-S100P (a urothelial marker) and anti-SV40 antibodies in 66 clinical urine samples exhibiting SV40 positivity and a decoy-cell appearance on Papanicolaou staining. The clinical voided urine samples included seven cases of renal transplantation, 47 cases of cancer therapy and 12 cases of non-neoplastic disease. SurePath(™) liquid-based cytology was used for the urine samples. BKV-infected cells were categorized as SV40(+)/S100P(+) and SV40 (+)/S100p(-). SV40(+)/S100P(-) cells were found in 55 cases (83.4%); nine cases (13.6%) carried both SV40(+)/S100P(-) and SV40(+)/S100P(+) cells. The former were identified as BKV infection in renal tubules and the latter in both the renal tubules and urothelial epithelia. The remaining two cases (3.0%) had only SV40(+)/S100P(+) cells of urothelial origin. Simultaneous immunostaining with anti-S100P and anti-SV40 is a useful method for determining the origin of BKV-infected cells in clinical urine samples from immunocompromised patients such as renal transplantation recipients. © 2014 John Wiley & Sons Ltd.

Single-cell immunofluorescence assay for terminal transferase: human leukaemic and non-leukaemic cells.

PubMed

Okamura, S; Crane, F; Jamal, N; Messner, H A; Mak, T W

1980-02-01

The characteristics of a single-cell immunofluorescence assay for terminal deoxynucleotidyl transferase (terminal transferase, TdT) is described. The data indicate that the single-cell immunofluorescence assay is highly efficient and specific for the detection of cells containing TdT. Using this assay, we have examined 124 marrow or peripheral-blood samples from 104 patients with or without haematological malignancies. Results indicate that TdT(+) cells from 6% to 100% were found in the following patients: 34/40 samples from patients with ALL at the time of diagnosis or during relapse; 2/3 patients with acute undifferentiated leukaemia; 2/3 patients with acute myelomonocytic leukaemia; 1/24 patients with acute myeloblastic leukaemia; 1/5 patients with chronic myelocytic leukaemia (CML) in blastic crisis; and 2/2 patients with diffuse lymphoblastic lymphoma. In contrast less than 1% of TdT(+) cells were found in 20 marrow or peripheral-blood samples from ALL patients in complete remission; 8 patients with CML in chronic phase; 2 patients with myeloma; 1 sample from a patient with Hodgkin's disease, peripheral-blood samples from 7 normal donors and marrow samples from 6 patients without haematological malignancies. TdT(+) cells were also found in association with cells with lymphoblast morphology. The TdT(+) cells in marrow were shown to be directly correlated with the percentage of morphological lymphoblasts, with a Spearman rank coefficient of 0·81, significant at a 0·001 level. In 2 longitudinal studies of 2 ALL patients with TdT(+) cells at diagnosis, the percentage TdT(+) cells also changed in parallel with the proportion of lymphoblasts. However, studies of 2 other patients with morphologically diagnosed ALL with < 1% TdT(+) cells at diagnosis also showed < 1% TdT(+) cells throughout the period studied, indicating a stable phenotype of blast cells in these patients. The single-cell immunofluorescence assay for TdT, which requires < 0·1% of the cells used in a conventional biochemical assay, is highly specific, and could provide a technically more efficient alternative for use in clinics as well as in experimental investigations of subpopulations of leukaemic and normal marrow cells.

Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA.

PubMed

Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takahashi, Shirushi; Kurosu, Akira; Takada, Aya; Saito, Kazuyuki

2016-05-01

A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real-time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples. © 2016 American Academy of Forensic Sciences.

Detection of living cells in stratospheric samples

NASA Astrophysics Data System (ADS)

Harris, Melanie J.; Wickramasinghe, N. C.; Lloyd, David; Narlikar, J. V.; Rajaratnam, P.; Turner, Michael P.; Al-Mufti, Shirwan; Wallis, Max K.; Ramadurai, S.; Hoyle, Fred

2002-02-01

Air samples collected aseptically over tropical India at various stratospheric altitudes ranging from 20 to 41 km using cryosampler assemblies carried on balloons flown from Hyderabad have shown evidence of living microbial cells. Unambiguous evidence of living cells came from examining micropore filters on which the samples were recovered with the use of voltage sensitive lipophilic dyes that could detect the presents of active cells. Clumps of viable cells were found at all altitudes using this technique, and this conclusion was found to be consistent with images obtained from electron microscopy. Since the 41 km sample was collected well above the local tropopause, a prima facie case for a space incidence of these microorganisms is established. Further work on culturing, PCR analysis and isotopic analysis is in progress.

Photoacoustic spectroscopy sample array vessels and photoacoustic spectroscopy methods for using the same

DOEpatents

Amonette, James E.; Autrey, S. Thomas; Foster-Mills, Nancy S.

2006-02-14

Methods and apparatus for simultaneous or sequential, rapid analysis of multiple samples by photoacoustic spectroscopy are disclosed. Particularly, a photoacoustic spectroscopy sample array vessel including a vessel body having multiple sample cells connected thereto is disclosed. At least one acoustic detector is acoustically positioned near the sample cells. Methods for analyzing the multiple samples in the sample array vessels using photoacoustic spectroscopy are provided.

Validation of a polymerase chain reaction aided transcript titration assay (PATTY) for topoisomerase II in lung cancer samples.

PubMed

Dingemans, A M; Van Ark-Otte, J; Smit, E F; Postmus, P E; Giaccone, G

This report describes the validation of a polymerase chain reaction aided transcript titration assay (PATTY) for tumor samples. The results obtained with the PATTY were compared to those of RNase protection in a set of 7 human lung cancer cell lines and in 23 non-small cell lung cancer samples derived from resected patients. Whereas between PATTY and RNase protection assay a good correlation was observed in the cell lines (r = 0.74, p = 0.057), no correlation was observed within the tumor samples (r = 0.06, p = 0.78). This was also the case when only tumors with a high percentage of tumor cells (> 90%) were selected. Although PATTY is a valuable tool to measure mRNA expression in cell lines, our results caution the use of PATTY in human tumor samples without proper validation. The possible causes of these results are discussed.

High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening.

PubMed

Young, Susan M; Curry, Mark S; Ransom, John T; Ballesteros, Juan A; Prossnitz, Eric R; Sklar, Larry A; Edwards, Bruce S

2004-03-01

HyperCyt, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with approximately 10,000 cells analyzed per reaction. Cell Ca(2+) responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.

Quantitation of the cellular content of saliva and buccal swab samples.

PubMed

Theda, Christiane; Hwang, Seo Hye; Czajko, Anna; Loke, Yuk Jing; Leong, Pamela; Craig, Jeffrey M

2018-05-02

Buccal swabs and saliva are the two most common oral sampling methods used for medical research. Often, these samples are used interchangeably, despite previous evidence that both contain buccal cells and blood leukocytes in different proportions. For some research, such as epigenetic studies, the cell types contributing to the analysis are highly relevant. We collected such samples from twelve children and twenty adults and, using Papanicolaou staining, measured the proportions of epithelial cells and leukocytes through microscopy. To our knowledge, no studies have compared cellular heterogeneity in buccal swab and saliva samples from adults and children. We confirmed that buccal swabs contained a higher proportion of epithelial cells than saliva and that children have a greater proportion of such cells in saliva compared to adults. At this level of resolution, buccal swabs and saliva contained similar epithelial cell subtypes. Gingivitis in children was associated with a higher proportion of leukocytes in saliva samples but not in buccal swabs. Compared to more detailed and costly methods such as flow cytometry or deconvolution methods used in epigenomic analysis, the procedure described here can serve as a simple and low-cost method to characterize buccal and saliva samples. Microscopy provides a low-cost tool to alert researchers to the presence of oral inflammation which may affect a subset of their samples. This knowledge might be highly relevant to their specific research questions, may assist with sample selection and thus might be crucial information despite the ability of data deconvolution methods to correct for cellular heterogeneity.

Design and optimization of reverse-transcription quantitative PCR experiments.

PubMed

Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael

2009-10-01

Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

Development of a novel method for amniotic fluid stem cell storage.

PubMed

Zavatti, Manuela; Beretti, Francesca; Casciaro, Francesca; Comitini, Giuseppina; Franchi, Fabrizia; Barbieri, Veronica; Bertoni, Laura; De Pol, Anto; La Sala, Giovanni B; Maraldi, Tullia

2017-08-01

Current procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. We compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. hAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Massively parallel digital transcriptional profiling of single cells

PubMed Central

Zheng, Grace X. Y.; Terry, Jessica M.; Belgrader, Phillip; Ryvkin, Paul; Bent, Zachary W.; Wilson, Ryan; Ziraldo, Solongo B.; Wheeler, Tobias D.; McDermott, Geoff P.; Zhu, Junjie; Gregory, Mark T.; Shuga, Joe; Montesclaros, Luz; Underwood, Jason G.; Masquelier, Donald A.; Nishimura, Stefanie Y.; Schnall-Levin, Michael; Wyatt, Paul W.; Hindson, Christopher M.; Bharadwaj, Rajiv; Wong, Alexander; Ness, Kevin D.; Beppu, Lan W.; Deeg, H. Joachim; McFarland, Christopher; Loeb, Keith R.; Valente, William J.; Ericson, Nolan G.; Stevens, Emily A.; Radich, Jerald P.; Mikkelsen, Tarjei S.; Hindson, Benjamin J.; Bielas, Jason H.

2017-01-01

Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients. PMID:28091601

Post-irradiation-examination of irradiated fuel outside the hot cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dawn E. Janney; Adam B. Robinson; Thomas P. O'Holleran

Because of their high radioactivity, irradiated fuels are commonly examined in a hot cell. However, the Idaho National Laboratory (INL) has recently investigated irradiated U-Mo-Al metallic fuel from the Reduced Enrichment for Research and Test Reactors (RERTR) project using a conventional unshielded scanning electron microscope outside a hot cell. This examination was possible because of a two-step sample-preparation approach in which a small volume of fuel was isolated in a hot cell and shielding was introduced during later stages of sample preparation. The resulting sample contained numerous sample-preparation artifacts but allowed analysis of microstructures from selected areas.

Influence of water content on the laser-induced breakdown spectroscopy analysis of human cell pellet

NASA Astrophysics Data System (ADS)

Moon, Youngmin; Han, Jung Hyun; Lee, Jong Jin; Jeong, Sungho

2015-12-01

The effects of water content change in a biological sample on the emission signal intensity and intensity ratio during LIBS analysis were investigated. To examine the effects of water content only avoiding matrix effects, a homogeneous human cell pellet consisting of cultured human immortalized keratinocyte cell only was used as the sample. LIBS spectra of the human cell pellet sample produced with a Q-switched Nd:YAG laser (λ = 532 nm, τ = 5 ns, top-hat profile) and a six-channel CCD spectrometer (spectral range = 187-1045 nm, spectral resolution = 0.1 nm) revealed that most of the emission lines observed from a tissue sample were also observable from the human cell pellet. The intensity and intensity ratio of the emission lines varied significantly as the water content of the human cell pellet was changed. It was found that a typically selected internal standard in LIBS analysis of biological samples such as carbon could produce inconsistent results, whereas the ratio of properly selected emission lines such as Mg(II) 280.270 nm and Ca(II) 396.847 nm was nearly independent of sample water content.

Isolation of circulating tumor cells in pancreatic cancer patients by immunocytochemical assay.

PubMed

Yang, Jing; Zhou, Ying; Zhao, Bin

2018-01-01

The patients diagnosed with pancreatic cancer have the possibilities of getting the cancer again even after resection. The tumor cells identified from blood can be related to different stages of tumor. In this study, we used an immunoassay to detect circulating tumor cells in blood and bone marrow samples. About 120 patients' blood and bone marrow samples were used in this study along with controls. The presence of tumor cells was evaluated with different stages of cancer classified by UICC. The survival rate at each stages of tumor was also analyzed. The tumor cells were isolated both in blood (29%) and bone marrow samples (25%). The prevalence of tumor cells increased with increase in stages of tumor in blood samples. The survival of the patients considerably related to different stages of tumor but it cannot be taken a parameter alone for the patients' survival. © 2017 Wiley Periodicals, Inc.

A novel approach to non-biased systematic random sampling: a stereologic estimate of Purkinje cells in the human cerebellum.

PubMed

Agashiwala, Rajiv M; Louis, Elan D; Hof, Patrick R; Perl, Daniel P

2008-10-21

Non-biased systematic sampling using the principles of stereology provides accurate quantitative estimates of objects within neuroanatomic structures. However, the basic principles of stereology are not optimally suited for counting objects that selectively exist within a limited but complex and convoluted portion of the sample, such as occurs when counting cerebellar Purkinje cells. In an effort to quantify Purkinje cells in association with certain neurodegenerative disorders, we developed a new method for stereologic sampling of the cerebellar cortex, involving calculating the volume of the cerebellar tissues, identifying and isolating the Purkinje cell layer and using this information to extrapolate non-biased systematic sampling data to estimate the total number of Purkinje cells in the tissues. Using this approach, we counted Purkinje cells in the right cerebella of four human male control specimens, aged 41, 67, 70 and 84 years, and estimated the total Purkinje cell number for the four entire cerebella to be 27.03, 19.74, 20.44 and 22.03 million cells, respectively. The precision of the method is seen when comparing the density of the cells within the tissue: 266,274, 173,166, 167,603 and 183,575 cells/cm3, respectively. Prior literature documents Purkinje cell counts ranging from 14.8 to 30.5 million cells. These data demonstrate the accuracy of our approach. Our novel approach, which offers an improvement over previous methodologies, is of value for quantitative work of this nature. This approach could be applied to morphometric studies of other similarly complex tissues as well.

A novel approach to non-biased systematic random sampling: A stereologic estimate of Purkinje cells in the human cerebellum

PubMed Central

Agashiwala, Rajiv M.; Louis, Elan D.; Hof, Patrick R.; Perl, Daniel P.

2010-01-01

Non-biased systematic sampling using the principles of stereology provides accurate quantitative estimates of objects within neuroanatomic structures. However, the basic principles of stereology are not optimally suited for counting objects that selectively exist within a limited but complex and convoluted portion of the sample, such as occurs when counting cerebellar Purkinje cells. In an effort to quantify Purkinje cells in association with certain neurodegenerative disorders, we developed a new method for stereologic sampling of the cerebellar cortex, involving calculating the volume of the cerebellar tissues, identifying and isolating the Purkinje cell layer and using this information to extrapolate non-biased systematic sampling data to estimate the total number of Purkinje cells in the tissues. Using this approach, we counted Purkinje cells in the right cerebella of four human male control specimens, aged 41, 67, 70 and 84 years, and estimated the total Purkinje cell number for the four entire cerebella to be 27.03, 19.74, 20.44 and 22.03 million cells, respectively. The precision of the method is seen when comparing the density of the cells within the tissue: 266,274, 173,166, 167,603 and 183,575 cells/cm3, respectively. Prior literature documents Purkinje cell counts ranging from 14.8 to 30.5 million cells. These data demonstrate the accuracy of our approach. Our novel approach, which offers an improvement over previous methodologies, is of value for quantitative work of this nature. This approach could be applied to morphometric studies of other similarly complex tissues as well. PMID:18725208

A new apparatus design for high temperature (up to 950°C) quasi-elastic neutron scattering in a controlled gaseous environment.

PubMed

al-Wahish, Amal; Armitage, D; al-Binni, U; Hill, B; Mills, R; Jalarvo, N; Santodonato, L; Herwig, K W; Mandrus, D

2015-09-01

A design for a sample cell system suitable for high temperature Quasi-Elastic Neutron Scattering (QENS) experiments is presented. The apparatus was developed at the Spallation Neutron Source in Oak Ridge National Lab where it is currently in use. The design provides a special sample cell environment under controlled humid or dry gas flow over a wide range of temperature up to 950 °C. Using such a cell, chemical, dynamical, and physical changes can be studied in situ under various operating conditions. While the cell combined with portable automated gas environment system is especially useful for in situ studies of microscopic dynamics under operational conditions that are similar to those of solid oxide fuel cells, it can additionally be used to study a wide variety of materials, such as high temperature proton conductors. The cell can also be used in many different neutron experiments when a suitable sample holder material is selected. The sample cell system has recently been used to reveal fast dynamic processes in quasi-elastic neutron scattering experiments, which standard probes (such as electrochemical impedance spectroscopy) could not detect. In this work, we outline the design of the sample cell system and present results demonstrating its abilities in high temperature QENS experiments.

Design and research of built-in sample cell with multiple optical reflections

NASA Astrophysics Data System (ADS)

Liu, Jianhui; Wang, Shuyao; Lv, Jinwei; Liu, Shuyang; Zhou, Tao; Jia, Xiaodong

2017-10-01

In the field of trace gas measurement, with the characteristics of high sensitivity, high selectivity and rapid detection, tunable diode laser absorption spectroscopy (TDLAS) is widely used in industrial process and trace gas pollution monitoring. Herriott cell is a common form of multiple reflections of the sample cell, the structure of the Herriott cell is relatively simple, which be used to application of trace gas absorption spectroscopy. In the pragmatic situation, the gas components are complicated, and the continuous testing process for a long time can lead to different degree of pollution and corrosion for the reflector in the sample cell. If the mirror is not cleaned up in time, it will have a great influence on the detection accuracy. In order to solve this problem in the process of harsh environment detection, this paper presents a design of the built-in sample cell to avoid the contact of gas and the mirror, thereby effectively reducing corrosion pollution. If there is optical pollution, direct replacement of the built-in optical sample cell can easily to be disassembled, and cleaned. The advantage of this design is long optical path, high precision, cost savings and so on.

Fluorescent Cell Barcoding for Multiplex Flow Cytometry

PubMed Central

Krutzik, Peter O.; Clutter, Matthew R.; Trejo, Angelica; Nolan, Garry P.

2011-01-01

Fluorescent Cell Barcoding (FCB) enables high throughput, i.e. high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10 to 100-fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines as well as primary peripheral blood samples. Important technical considerations such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis are discussed. PMID:21207359

Enhanced cell adhesion on severe peened-plasma nitrided 316L stainless steel

NASA Astrophysics Data System (ADS)

Jayalakshmi, M.; Bhat, Badekai Ramachandra; Bhat, K. Udaya

2018-04-01

Plasma nitriding is an effective technique to enhance the wear resistance of austenitic stainless steels. Recently, severe surface deformation techniques are extensively used prior to nitriding to enhance diffusion kinetics. In the present study, AISI 316L austenitic stainless steel is subjected to peening-nitriding duplex treatment and biocompatibility of treated surfaces is assessed through adhesion of the fibroblast cells. Three-fold increase in the surface microhardness is observed from the un-peened sample to the peened-nitrided sample; with severe peened sample showing intermediate hardness. Similar trend is observed in the number of the fibroblast cells attached to the sample surface. Spreading of some of the fibroblast cells is observed on the sample subjected to duplex treatment; while the other two samples showed only the spindle shaped fibroblasts. Combined influence of surface nanocrystallization and presence of nitride layer is responsible for the improved biocompatibility.

Tuberculosis in Pap samples with emphasis on LBC: Caught only when thought.

PubMed

Bharani, Vani; Gupta, Nalini; Suri, Vanita; Rajwanshi, Arvind

2018-05-01

Despite being a commonly encountered infection, the clinical diagnosis of tuberculosis of the uterine cervix is elusive. Though a straightforward diagnosis on tissue sections, identification of typical features of tubercular infection on cervical Pap samples is challenging. In our experience, the infrequent pale staining collections of epithelioid cells are difficult to pick up on Pap stained smears, particularly LBC samples. In this series, 2 of the three samples were reported as atypical squamous cells of undetermined significance while 1 was reported as inflammatory at the initial diagnosis. Scattered Langhans' type giant cells may be seen as a subtle clue which should prompt the search for epithelioid cell granulomas. These cases may have a mass lesion clinically while no obvious signs of malignancy on the cervical samples. © 2017 Wiley Periodicals, Inc.

Performance evaluation of the Abbott CELL-DYN Ruby and the Sysmex XT-2000i haematology analysers.

PubMed

Leers, M P G; Goertz, H; Feller, A; Hoffmann, J J M L

2011-02-01

Two mid-range haematology analysers (Abbott CELL-DYN Ruby and Sysmex XT-2000i) were evaluated to determine their analytical performance and workflow efficiency in the haematology laboratory. In total 418 samples were processed for determining equivalence of complete blood count (CBC) measurements, and 100 for reticulocyte comparison. Blood smears served for assessing the agreement of the differential counts. Inter-instrument agreement for most parameters was good although small numbers of discrepancies were observed. Systematic biases were found for mean cell volume, reticulocytes, platelets and mean platelet volume. CELL-DYN Ruby WBC differentials were obtained with all samples while the XT-2000i suppressed differentials partially or completely in 13 samples (3.1%). WBC subpopulation counts were otherwise in good agreement with no major outliers. Following first-pass CBC/differential analysis, 88 (21%) of XT-2000i samples required further analyser processing compared to 18 (4.3%) for the CELL-DYN Ruby. Smear referrals for suspected WBC/nucleated red blood cells and platelet abnormalities were indicated for 106 (25.4%) and 95 (22.7%) of the XT-2000i and CELL-DYN Ruby samples respectively. Flagging efficiencies for both analysers were found to be similar. The Sysmex XT-2000i and Abbott CELL-DYN Ruby analysers have broadly comparable analytical performance, but the CELL-DYN Ruby showed superior first-pass efficiency. © 2010 Blackwell Publishing Ltd.

The contribution of cell blocks in the diagnosis of mediastinal masses and hilar adenopathy samples from echobronchoscopy.

PubMed

Lourido-Cebreiro, Tamara; Leiro-Fernández, Virginia; Tardio-Baiges, Antoni; Botana-Rial, Maribel; Núñez-Delgado, Manuel; Álvarez-Martín, M Jesús; Fernández-Villar, Alberto

2014-07-01

Cell block material from puncture can be obtained with endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in many cases. The aim of this study was to analyse the value of additional information from cell blocks obtained with EBUS-TBNA samples from mediastinal and hilar lymph nodes and masses. Review of pathology reports with a specific diagnosis obtained from EBUS-TBNA samples of mediastinal or hilar lesions, prospectively obtained over a two-year period. The generation of cell blocks from cytology needle samples, the contribution to morphological diagnosis, and the possible use of samples for immunohistochemistry were analysed. One hundred and twenty-nine samples corresponding to 110 patients were reviewed. The diagnosis was lung cancer in 81% of cases, extrapulmonary carcinoma in 10%, sarcoidosis in 4%, lymphoma in 2.7%, and tuberculosis in 0.9%. Cell blocks could be obtained in 72% of cases. Immunohistochemistry studies on the cell blocks were significantly easier to perform than on conventional smears (52.6% vs. 14%, P<.0001). In 4cases, the cell block provided an exclusive morphological diagnosis (3sarcoidosis and one metastasis from prostatic carcinoma) and in 3carcinomas, subtype and origin could be identified. Exclusive diagnoses from the cell block were significantly more frequent in benign disease than in malignant disease (25% vs 0.9%, P=.002). Cell blocks were obtained from 72% of EBUS-TBNA diagnostic procedures. The main contributions of cell blocks to pathology examinations were the possibility of carrying out immunohistochemical staining for the better classification of neoplasms, especially extrapulmonary metastatic tumours, and the improved diagnosis of benign lesions. Copyright © 2013 SEPAR. Published by Elsevier Espana. All rights reserved.

Targeting of colony-stimulating factor 1 receptor (CSF1R) in the CLL microenvironment yields antineoplastic activity in primary patient samples.

PubMed

Edwards V, David K; Sweeney, David Tyler; Ho, Hibery; Eide, Christopher A; Rofelty, Angela; Agarwal, Anupriya; Liu, Selina Qiuying; Danilov, Alexey V; Lee, Patrice; Chantry, David; McWeeney, Shannon K; Druker, Brian J; Tyner, Jeffrey W; Spurgeon, Stephen E; Loriaux, Marc M

2018-05-15

In many malignancies, the tumor microenvironment includes CSF1R-expressing supportive monocyte/macrophages that promote tumor cell survival. For chronic lymphocytic leukemia (CLL), these supportive monocyte/macrophages are known as nurse-like cells (NLCs), although the potential effectiveness of selective small-molecule inhibitors of CSF1R against CLL is understudied. Here, we demonstrate the preclinical activity of two inhibitors of CSF1R, GW-2580 and ARRY-382, in primary CLL patient samples. We observed at least 25% of CLL samples showed sub-micromolar sensitivity to CSF1R inhibitors. This sensitivity was observed in samples with varying genetic and clinical backgrounds, although higher white cell count and monocyte cell percentage was associated with increased sensitivity. Depleting CD14-expressing monocytes preferentially decreased viability in samples sensitive to CSF1R inhibitors, and treating samples with CSF1R inhibitors eliminated the presence of NLCs in long-term culture conditions. These results indicate that CSF1R small-molecule inhibitors target CD14-expressing monocytes in the CLL microenvironment, thereby depriving leukemia cells of extrinsic support signals. In addition, significant synergy was observed combining CSF1R inhibitors with idelalisib or ibrutinib, two current CLL therapies that disrupt tumor cell intrinsic B-cell receptor signaling. These findings support the concept of simultaneously targeting supportive NLCs and CLL cells and demonstrate the potential clinical utility of this combination.

Availability of a fetal goat tongue cell line ZZ-R 127 for isolation of Foot-and-mouth disease virus (FMDV) from clinical samples collected from animals experimentally infected with FMDV.

PubMed

Fukai, Katsuhiko; Onozato, Hiroyuki; Kitano, Rie; Yamazoe, Reiko; Morioka, Kazuki; Yamada, Manabu; Ohashi, Seiichi; Yoshida, Kazuo; Kanno, Toru

2013-11-01

The availability of the fetal goat tongue cell line ZZ-R 127 for the isolation of Foot-and-mouth disease virus (FMDV) has not been evaluated using clinical samples other than epithelial suspensions. Therefore, in the current study, the availability of ZZ-R 127 cells for the isolation of FMDV was evaluated using clinical samples (e.g., sera, nasal swabs, saliva, feces, and oropharyngeal fluids) collected from animals experimentally infected with an FMDV isolate. Virus isolation rates for the ZZ-R 127 cells were statistically higher than those for the porcine kidney cell line (IB-RS-2) in experimental infections using cattle, goats, and pigs (P < 0.01). Virus titers in the ZZ-R 127 cells were also statistically higher than those in the IB-RS-2 cells. The availability of ZZ-R 127 cells for the isolation of FMDV not only from epithelial suspensions but also from other clinical samples was confirmed in the current study.

Pulsed Direct Current Electrospray: Enabling Systematic Analysis of Small Volume Sample by Boosting Sample Economy.

PubMed

Wei, Zhenwei; Xiong, Xingchuang; Guo, Chengan; Si, Xingyu; Zhao, Yaoyao; He, Muyi; Yang, Chengdui; Xu, Wei; Tang, Fei; Fang, Xiang; Zhang, Sichun; Zhang, Xinrong

2015-11-17

We had developed pulsed direct current electrospray ionization mass spectrometry (pulsed-dc-ESI-MS) for systematically profiling and determining components in small volume sample. Pulsed-dc-ESI utilized constant high voltage to induce the generation of single polarity pulsed electrospray remotely. This method had significantly boosted the sample economy, so as to obtain several minutes MS signal duration from merely picoliter volume sample. The elongated MS signal duration enable us to collect abundant MS(2) information on interested components in a small volume sample for systematical analysis. This method had been successfully applied for single cell metabolomics analysis. We had obtained 2-D profile of metabolites (including exact mass and MS(2) data) from single plant and mammalian cell, concerning 1034 components and 656 components for Allium cepa and HeLa cells, respectively. Further identification had found 162 compounds and 28 different modification groups of 141 saccharides in a single Allium cepa cell, indicating pulsed-dc-ESI a powerful tool for small volume sample systematical analysis.

Crystal-Chemical Analysis Martian Minerals in Gale Crater

NASA Technical Reports Server (NTRS)

Morrison, S. M.; Downs, R. T.; Blake, D. F.; Bish, D. L.; Ming, D. W.; Morris, R. V.; Yen, A. S.; Chipera, S. J.; Treiman, A. H.; Vaniman, D. T.;

Scalable multi-sample single-cell data analysis by Partition-Assisted Clustering and Multiple Alignments of Networks

PubMed Central

Samusik, Nikolay; Wang, Xiaowei; Guan, Leying; Nolan, Garry P.

2017-01-01

Mass cytometry (CyTOF) has greatly expanded the capability of cytometry. It is now easy to generate multiple CyTOF samples in a single study, with each sample containing single-cell measurement on 50 markers for more than hundreds of thousands of cells. Current methods do not adequately address the issues concerning combining multiple samples for subpopulation discovery, and these issues can be quickly and dramatically amplified with increasing number of samples. To overcome this limitation, we developed Partition-Assisted Clustering and Multiple Alignments of Networks (PAC-MAN) for the fast automatic identification of cell populations in CyTOF data closely matching that of expert manual-discovery, and for alignments between subpopulations across samples to define dataset-level cellular states. PAC-MAN is computationally efficient, allowing the management of very large CyTOF datasets, which are increasingly common in clinical studies and cancer studies that monitor various tissue samples for each subject. PMID:29281633

Improved efficiency of InGaN/GaN-based multiple quantum well solar cells by reducing contact resistance

NASA Astrophysics Data System (ADS)

Song, Jun-Hyuk; Oh, Joon-Ho; Shim, Jae-Phil; Min, Jung-Hong; Lee, Dong-Seon; Seong, Tae-Yeon

2012-08-01

We report on the improvement in the performance of InGaN/GaN multi-quantum well-based solar cells by the introduction of a Cu-doped indium oxide (CIO) layer at the interface between indium tin oxide (ITO) p-electrode and p-GaN. The solar cell fabricated with the 3 nm-sample exhibits an external quantum efficiency of 29.8% (at a peak wavelength of 376 nm) higher than those (25.2%) of the cell with the ITO-only sample. The use of the 3-nm-thick CIO layer gives higher short circuit current density (0.72 mA/cm2) and fill factor (78.85%) as compared to those (0.65 mA/cm2 and 74.08%) of the ITO only sample. Measurements show that the conversion efficiency of the solar cells with the ITO-only sample and the 3 nm-sample is 1.12% and 1.30%, respectively. Based on their electrical and optical properties, the dependence of the CIO interlayer thickness on the efficiency of solar cells is discussed.

Identification of Highly Methylated Genes across Various Types of B-Cell Non-Hodgkin Lymphoma

PubMed Central

Bethge, Nicole; Honne, Hilde; Hilden, Vera; Trøen, Gunhild; Eknæs, Mette; Liestøl, Knut; Holte, Harald; Delabie, Jan; Smeland, Erlend B.; Lind, Guro E.

2013-01-01

Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n = 480) when compared to normal B cells (n = 5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n = 42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n = 25), confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients. PMID:24260260

Do weather conditions correlate with findings in failed, provision-filled nest cells of Megachile rotundata (Hymenoptera: Megachilidae) in western North America?

PubMed

Pitts-Singer, Theresa L; James, Rosalind R

2008-06-01

Cavity-nesting alfalfa leafcutting bees, Megachile rotundata (F.) (Hymenoptera: Megachilidae), are excellent pollinators of alfalfa, Medicago savita L., for seed production. In commercial settings, artificial cavities are placed in field domiciles for nesting and, thereby, bee populations are sustained for future use. For this study, cells from leafcutting bee nests were collected in late summer from commercial seed fields. Over 3 yr (2003-2005), 39 samples in total of approximately equal to 1,000 cells each were taken from several northwestern U.S. states and from Manitoba, Canada. X-radiography of 500 cells from each sample was used to identify "pollen balls" (i.e., cells in which the pollen-nectar provision remained, but the egg or larva, if present, was not detectable on an x-radiograph). Most U.S. samples seemed to have higher proportions of pollen ball cells than Manitoba samples. Pollen ball cells were dissected to determine the moisture condition of the mass provision and true contents of each cell. Most pollen ball cells from Manitoba samples contained fungus, the frequency of which was positively correlated with cool, wet weather. In the United States, most pollen ball cells had moist provisions, and many of them lacked young brood. Correlation analysis revealed that pollen ball cells occurred in greater proportions in fields with more hot days (above 38 degrees C). Broodless pollen ball cells occurred in greater proportions under cool conditions, but dead small larvae (second-third instars) seemed to occur in greater proportions under hot conditions. Pollen ball cells with unhatched eggs and first instars (in the chorion) occurred in lesser proportions under hot conditions.

Detection of cytomegalovirus DNA in fecal samples as a method for CMV enterocolitis diagnosis after allogeneic stem cell transplantation.

PubMed

Zavrelova, Alzbeta; Radocha, Jakub; Pliskova, Lenka; Paterova, Pavla; Vejrazkova, Eva; Cyrany, Jiri; Gabalec, Filip; Podhola, Miroslav; Zak, Pavel

2018-05-16

Cytomegalovirus enterocolitis is a rare but potentially life threatening complication after allogeneic stem cell transplantation. Its early diagnosis and treatment are essential for a successful outcome. To determine the potential benefit of fecal CMV DNA detection in the diagnosis of CMV colitis among stem cell transplant recipients. Biopsies from the lower gastrointestinal tract, taken during 69 episodes of diarrhea, were compared with fecal samples previously examined for CMV DNA in 45 patients after allogeneic stem cell transplantation. Six confirmed cases of CMV colitis were observed, with 16 out of 69 (23%) fecal samples proving positive for CMV DNA. Only one positive sample correlated with histologically confirmed CMV colitis, and 15 samples were evaluated as false positive. These results provide a 16.7% sensitivity and 76.2% specificity in the diagnosis of CMV enterocolitis. The examination of fecal samples for the presence of CMV DNA has very low potential in the diagnosis of CMV enterocolitis after allogeneic stem cell transplantation; therefore, a biopsy of the gastrointestinal mucosa is still warranted for correct diagnosis.

A new x-ray interface and surface scattering environmental cell design for in situ studies of radioactive and atmosphere-sensitive samples.

PubMed

Schmidt, M; Eng, P J; Stubbs, J E; Fenter, P; Soderholm, L

2011-07-01

We present a novel design of a purpose-built, portable sample cell for in situ x-ray scattering experiments of radioactive or atmosphere sensitive samples. The cell has a modular design that includes two independent layers of containment that are used simultaneously to isolate the sensitive samples. Both layers of containment can be flushed with an inert gas, thus serving a double purpose as containment of radiological material (either as a solid sample or as a liquid phase) and in separating reactive samples from the ambient atmosphere. A remote controlled solution flow system is integrated into the containment system that allows sorption experiments to be performed on the diffractometer. The cell's design is discussed in detail and we demonstrate the cell's performance by presenting first results of crystal truncation rod measurements. The results were obtained from muscovite mica single crystals reacted with 1 mM solutions of Th(IV) with 0.1 M NaCl background electrolyte. Data were obtained in specular as well as off-specular geometry.

Hall Effect Thruster Plume Contamination and Erosion Study

NASA Technical Reports Server (NTRS)

Jaworske, Donald A.

2000-01-01

The objective of the Hall effect thruster plume contamination and erosion study was to evaluate the impact of a xenon ion plume on various samples placed in the vicinity of a Hall effect thruster for a continuous 100 hour exposure. NASA Glenn Research Center was responsible for the pre- and post-test evaluation of three sample types placed around the thruster: solar cell cover glass, RTV silicone, and Kapton(R). Mass and profilometer), were used to identify the degree of deposition and/or erosion on the solar cell cover glass, RTV silicone, and Kapton@ samples. Transmittance, reflectance, solar absorptance, and room temperature emittance were used to identify the degree of performance degradation of the solar cell cover glass samples alone. Auger spectroscopy was used to identify the chemical constituents found on the surface of the exposed solar cell cover glass samples. Chemical analysis indicated some boron nitride contamination on the samples, from boron nitride insulators used in the body of the thruster. However, erosion outweighted contamination. All samples exhibited some degree of erosion. with the most erosion occurring near the centerline of the plume and the least occurring at the +/- 90 deg positions. For the solar cell cover glass samples, erosion progressed through the antireflective coating and into the microsheet glass itself. Erosion occurred in the solar cell cover glass, RTV silicone and Kapton(R) at different rates. All optical properties changed with the degree of erosion, with solar absorptance and room temperature emittance increasing with erosion. The transmittance of some samples decreased while the reflectance of some samples increased and others decreased. All results are consistent with an energetic plume of xenon ions serving as a source for erosion.

A New Method for Estimating Bacterial Abundances in Natural Samples using Sublimation

NASA Technical Reports Server (NTRS)

Glavin, Daniel P.; Cleaves, H. James; Schubert, Michael; Aubrey, Andrew; Bada, Jeffrey L.

2004-01-01

We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert were heated to a temperature of 500 C for several seconds under reduced pressure. The sublimate was collected on a cold finger and the amount of adenine released from the samples then determined by high performance liquid chromatography (HPLC) with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from approx. l0(exp 5) to l0(exp 9) E. coli cell equivalents per gram. For most of these samples, the sublimation based cell counts were in agreement with total bacterial counts obtained by traditional DAPI staining. The simplicity and robustness of the sublimation technique compared to the DAPI staining method makes this approach particularly attractive for use by spacecraft instrumentation. NASA is currently planning to send a lander to Mars in 2009 in order to assess whether or not organic compounds, especially those that might be associated with life, are present in Martian surface samples. Based on our analyses of the Atacama Desert soil samples, several million bacterial cells per gam of Martian soil should be detectable using this sublimation technique.

Evaluation of p16 hypermethylation in oral submucous fibrosis: A quantitative and comparative analysis in buccal cells and saliva using real-time methylation-specific polymerase chain reaction.

PubMed

Kaliyaperumal, Subadra; Sankarapandian, Sathasivasubramanian

2016-01-01

The aim of this study was to quantitatively investigate the hypermethylation of p16 gene in buccal cells and saliva of oral submucous fibrosis (OSMF) patients using real-time quantitative methylation-specific polymerase chain reaction (PCR) and to compare the values of two methods. A total of 120 samples were taken from 60 subjects selected for this study, of which 30 were controls and 30 patients were clinically and histopathologically diagnosed with OSMF. In both groups, two sets of samples were collected, one directly from the buccal cells through cytobrush technique and the other through salivary rinse. We analyzed the samples for the presence of p16 hypermethylation using quantitative real-time PCR. In OSMF, the hypermethylation status of p16 in buccal cells was very high (93.3%) and in salivary samples, it was partially methylated (50%). However, no hypermethylation was found in controls suggesting that significant quantity of p16 hypermethylation was present in buccal cells and saliva in OSMF. This study indicates that buccal cell sampling may be a better method for evaluation than the salivary samples. It signifies that hypermethylation of p16 is an important factor to be considered in epigenetic alterations of normal cells to oral precancer, i.e. OSMF.

Repeated sampling of genes from a single cell - implications for gravitropism research

NASA Astrophysics Data System (ADS)

Scherp, P.; Hasenstein, K. H.

The need for repeated but independent extractions of mRNA from single cells and plant tissues prompted the development of Solid Phase Gene Extraction (SPGE, patent pending). Oligo dT18 coated glass needles hybridize during a 2 to 3 min sampling time with the poly A+ mRNA. The needle is withdrawn and can be used directly for RT-PCR. Because of the small probe size, no cytoplasm is lost and repeated sampling of the same cell is possible. SPGE of Chara rhizoids and internodal cells showed fluctuations of type and quantity of mRNA in specific areas of the cytoplasm of rhizoids and time-dependent gene expression in internodal cells as a function of light/dark intervals. Despite extensive cytoplasmic streaming, mRNA-samples taken in the vicinity of the nucleus revealed a higher variability than the distal ends of the cell. In rhizoids, the mRNA/cDNA varied between the different zones of cytoplasm. In Arabidopsis, we isolated cDNA species from root tips, shoots and leaves and determined their sequences. Growth studies on SPGE-sampled individuals showed that after a short recovery period, all sampled plants resumed growth with normal growth rates and graviresponse. The data indicate that SPGE is a powerful method to study gene expression in single cells and in tissues of higher plants with high spatial and temporal resolution. Supported by NASA: NAG 2-1423

Cost-effective and Rapid Blood Analysis on a Cell-phone

PubMed Central

Zhu, Hongying; Sencan, Ikbal; Wong, Justin; Dimitrov, Stoyan; Tseng, Derek; Nagashima, Keita; Ozcan, Aydogan

2013-01-01

We demonstrate a compact and cost-effective imaging cytometry platform installed on a cell-phone for the measurement of the density of red and white blood cells as well as hemoglobin concentration in human blood samples. Fluorescent and bright-field images of blood samples are captured using separate optical attachments to the cell-phone and are rapidly processed through a custom-developed smart application running on the phone for counting of blood cells and determining hemoglobin density. We evaluated the performance of this cell-phone based blood analysis platform using anonymous human blood samples and achieved comparable results to a standard bench-top hematology analyser. Test results can either be stored on the cell-phone memory or be transmitted to a central server, providing remote diagnosis opportunities even in field settings. PMID:23392286

Cost-effective and rapid blood analysis on a cell-phone.

PubMed

Zhu, Hongying; Sencan, Ikbal; Wong, Justin; Dimitrov, Stoyan; Tseng, Derek; Nagashima, Keita; Ozcan, Aydogan

2013-04-07

We demonstrate a compact and cost-effective imaging cytometry platform installed on a cell-phone for the measurement of the density of red and white blood cells as well as hemoglobin concentration in human blood samples. Fluorescent and bright-field images of blood samples are captured using separate optical attachments to the cell-phone and are rapidly processed through a custom-developed smart application running on the phone for counting of blood cells and determining hemoglobin density. We evaluated the performance of this cell-phone based blood analysis platform using anonymous human blood samples and achieved comparable results to a standard bench-top hematology analyser. Test results can either be stored on the cell-phone memory or be transmitted to a central server, providing remote diagnosis opportunities even in field settings.

Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.

PubMed

Shin, Saeam; Kim, Juwon; Kim, Yoonjung; Cho, Sun-Mi; Lee, Kyung-A

2017-10-26

EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

A novel portable filtration system for sampling and concentration of microorganisms: Demonstration on marine microalgae with subsequent quantification using IC-NASBA.

PubMed

Loukas, Christos-Moritz; Mowlem, Matthew C; Tsaloglou, Maria-Nefeli; Green, Nicolas G

2018-05-01

This paper presents a novel portable sample filtration/concentration system, designed for use on samples of microorganisms with very low cell concentrations and large volumes, such as water-borne parasites, pathogens associated with faecal matter, or toxic phytoplankton. The example application used for demonstration was the in-field collection and concentration of microalgae from seawater samples. This type of organism is responsible for Harmful Algal Blooms (HABs), an example of which is commonly referred to as "red tides", which are typically the result of rapid proliferation and high biomass accumulation of harmful microalgal species in the water column or at the sea surface. For instance, Karenia brevis red tides are the cause of aquatic organism mortality and persistent blooms may cause widespread die-offs of populations of other organisms including vertebrates. In order to respond to, and adequately manage HABs, monitoring of toxic microalgae is required and large-volume sample concentrators would be a useful tool for in situ monitoring of HABs. The filtering system presented in this work enables consistent sample collection and concentration from 1 L to 1 mL in five minutes, allowing for subsequent benchtop sample extraction and analysis using molecular methods such as NASBA and IC-NASBA. The microalga Tetraselmis suecica was successfully detected at concentrations ranging from 2 × 10 5  cells/L to 20 cells/L. Karenia brevis was also detected and quantified at concentrations between 10 cells/L and 10 6  cells/L. Further analysis showed that the filter system, which concentrates cells from very large volumes with consequently more reliable sampling, produced samples that were more consistent than the independent non-filtered samples (benchtop controls), with a logarithmic dependency on increasing cell numbers. This filtering system provides simple, rapid, and consistent sample collection and concentration for further analysis, and could be applied to a wide range of different samples and target organisms in situations lacking laboratories. Copyright © 2018. Published by Elsevier B.V.

Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells.

PubMed

Wolffs, Petra; Norling, Börje; Rådström, Peter

2005-03-01

Real-time PCR technology is increasingly used for detection and quantification of pathogens in food samples. A main disadvantage of nucleic acid detection is the inability to distinguish between signals originating from viable cells and DNA released from dead cells. In order to gain knowledge concerning risks of false-positive results due to detection of DNA originating from dead cells, quantitative PCR (qPCR) was used to investigate the degradation kinetics of free DNA in four types of meat samples. Results showed that the fastest degradation rate was observed (1 log unit per 0.5 h) in chicken homogenate, whereas the slowest rate was observed in pork rinse (1 log unit per 120.5 h). Overall results indicated that degradation occurred faster in chicken samples than in pork samples and faster at higher temperatures. Based on these results, it was concluded that, especially in pork samples, there is a risk of false-positive PCR results. This was confirmed in a quantitative study on cell death and signal persistence over a period of 28 days, employing three different methods, i.e. viable counts, direct qPCR, and finally floatation, a recently developed discontinuous density centrifugation method, followed by qPCR. Results showed that direct qPCR resulted in an overestimation of up to 10 times of the amount of cells in the samples compared to viable counts, due to detection of DNA from dead cells. However, after using floatation prior to qPCR, results resembled the viable count data. This indicates that by using of floatation as a sample treatment step prior to qPCR, the risk of false-positive PCR results due to detection of dead cells, can be minimized.

Microfluidic cell isolation technology for drug testing of single tumor cells and their clusters.

PubMed

Bithi, Swastika S; Vanapalli, Siva A

2017-02-02

Drug assays with patient-derived cells such as circulating tumor cells requires manipulating small sample volumes without loss of rare disease-causing cells. Here, we report an effective technology for isolating and analyzing individual tumor cells and their clusters from minute sample volumes using an optimized microfluidic device integrated with pipettes. The method involves using hand pipetting to create an array of cell-laden nanoliter-sized droplets immobilized in a microfluidic device without loss of tumor cells during the pipetting process. Using this technology, we demonstrate single-cell analysis of tumor cell response to the chemotherapy drug doxorubicin. We find that even though individual tumor cells display diverse uptake profiles of the drug, the onset of apoptosis is determined by accumulation of a critical intracellular concentration of doxorubicin. Experiments with clusters of tumor cells compartmentalized in microfluidic drops reveal that cells within a cluster have higher viability than their single-cell counterparts when exposed to doxorubicin. This result suggests that circulating tumor cell clusters might be able to better survive chemotherapy drug treatment. Our technology is a promising tool for understanding tumor cell-drug interactions in patient-derived samples including rare cells.

Novel flowcytometry-based approach of malignant cell detection in body fluids using an automated hematology analyzer

PubMed Central

Tabe, Yoko; Takemura, Hiroyuki; Kimura, Konobu; Takahashi, Toshihiro; Yang, Haeun; Tsuchiya, Koji; Konishi, Aya; Uchihashi, Kinya; Horii, Takashi; Ohsaka, Akimichi

2018-01-01

Morphological microscopic examinations of nucleated cells in body fluid (BF) samples are performed to screen malignancy. However, the morphological differentiation is time-consuming and labor-intensive. This study aimed to develop a new flowcytometry-based gating analysis mode “XN-BF gating algorithm” to detect malignant cells using an automated hematology analyzer, Sysmex XN-1000. XN-BF mode was equipped with WDF white blood cell (WBC) differential channel. We added two algorithms to the WDF channel: Rule 1 detects larger and clumped cell signals compared to the leukocytes, targeting the clustered malignant cells; Rule 2 detects middle sized mononuclear cells containing less granules than neutrophils with similar fluorescence signal to monocytes, targeting hematological malignant cells and solid tumor cells. BF samples that meet, at least, one rule were detected as malignant. To evaluate this novel gating algorithm, 92 various BF samples were collected. Manual microscopic differentiation with the May-Grunwald Giemsa stain and WBC count with hemocytometer were also performed. The performance of these three methods were evaluated by comparing with the cytological diagnosis. The XN-BF gating algorithm achieved sensitivity of 63.0% and specificity of 87.8% with 68.0% for positive predictive value and 85.1% for negative predictive value in detecting malignant-cell positive samples. Manual microscopic WBC differentiation and WBC count demonstrated 70.4% and 66.7% of sensitivities, and 96.9% and 92.3% of specificities, respectively. The XN-BF gating algorithm can be a feasible tool in hematology laboratories for prompt screening of malignant cells in various BF samples. PMID:29425230

Novel flowcytometry-based approach of malignant cell detection in body fluids using an automated hematology analyzer.

PubMed

Ai, Tomohiko; Tabe, Yoko; Takemura, Hiroyuki; Kimura, Konobu; Takahashi, Toshihiro; Yang, Haeun; Tsuchiya, Koji; Konishi, Aya; Uchihashi, Kinya; Horii, Takashi; Ohsaka, Akimichi

2018-01-01

Morphological microscopic examinations of nucleated cells in body fluid (BF) samples are performed to screen malignancy. However, the morphological differentiation is time-consuming and labor-intensive. This study aimed to develop a new flowcytometry-based gating analysis mode "XN-BF gating algorithm" to detect malignant cells using an automated hematology analyzer, Sysmex XN-1000. XN-BF mode was equipped with WDF white blood cell (WBC) differential channel. We added two algorithms to the WDF channel: Rule 1 detects larger and clumped cell signals compared to the leukocytes, targeting the clustered malignant cells; Rule 2 detects middle sized mononuclear cells containing less granules than neutrophils with similar fluorescence signal to monocytes, targeting hematological malignant cells and solid tumor cells. BF samples that meet, at least, one rule were detected as malignant. To evaluate this novel gating algorithm, 92 various BF samples were collected. Manual microscopic differentiation with the May-Grunwald Giemsa stain and WBC count with hemocytometer were also performed. The performance of these three methods were evaluated by comparing with the cytological diagnosis. The XN-BF gating algorithm achieved sensitivity of 63.0% and specificity of 87.8% with 68.0% for positive predictive value and 85.1% for negative predictive value in detecting malignant-cell positive samples. Manual microscopic WBC differentiation and WBC count demonstrated 70.4% and 66.7% of sensitivities, and 96.9% and 92.3% of specificities, respectively. The XN-BF gating algorithm can be a feasible tool in hematology laboratories for prompt screening of malignant cells in various BF samples.

Chromatographic measurements of hemoglobin A2 in blood samples that contain sickle hemoglobin.

PubMed

Shokrani, M; Terrell, F; Turner, E A; Aguinaga, M D

2000-04-01

In the sickle cell syndromes, Hb A2 measurements aid in the differential diagnosis of sickle cell anemia from sickle-beta-thalassemia. The purpose of this study is to assess the Hb A2 levels in samples containing sickle hemoglobin (Hb S) by the use of an automated high performance liquid chromatography system (HPLC-Variant beta-thalassemia Short Program). The blood samples analyzed were from individuals of African descent living in the state of Tennessee who had either sickle cell trait (Hb AS), sickle cell disease (Hb SS), or sickle cell-hemoglobin C disease (Hb SC). Interestingly, the Hb A2 levels determined by HPLC were found elevated in samples containing Hb S. The Hb A2 mean in Hb AS samples (n=146) is 4.09% (SD +/- 0.42, range 2.20 to 5.20%); in Hb SS samples (n=33) it is 3.90% (SD +/- 1.08, range 0.60 to 5.90%); and in Hb SC samples (n=27) it is 4.46% (SD +/- 0.70, range 2.30 to 5.91%). The Hb A2 mean by HPLC in normal individuals (Hb AA, n=70) is 2.57% (SD +/- 0.25, range 2.1 to 3.0%), and the Hb A2 range in beta-thalassemia carriers is 4 to 9%. Our results show that the Hb A2 levels in Hb S-containing samples partially overlap with those expected from beta-thalassemia carriers. The hemoglobinopathy laboratory should be aware of this apparent elevation in Hb A2 levels determined by HPLC in individuals carrying Hb S. Other factors, such as family history and clinical symptoms, should be taken into account before a diagnosis of sickle cell trait, sickle-beta-thalassemia, or sickle cell anemia is made.

Characterization of the Tissue and Stromal Cell Components of Micro-Superficial Enhanced Fluid Fat Injection (Micro-SEFFI) for Facial Aging Treatment.

PubMed

Rossi, Martina; Roda, Barbara; Zia, Silvia; Vigliotta, Ilaria; Zannini, Chiara; Alviano, Francesco; Bonsi, Laura; Zattoni, Andrea; Reschiglian, Pierluigi; Gennai, Alessandro

2018-06-14

New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for regenerative purposes, the cellular content of the sample should be considered. To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples. Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector ®, an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays. Microscopic and quality analyses of micro-SEFFI samples by Celector® confirmed the high fluidity and sample cellular composition in terms of red blood cell contamination, the presence of cell aggregates and extracellular matrix fragments. ASCs were isolated from adipose tissue harvested using SEFFI and micro-SEFFI systems. These cells were demonstrated to have a good proliferation rate and differentiation potential towards mesenchymal lineages. Despite the small sizes and low cellularity observed in micro-SEFFI-derived tissue, we were able to isolate stem cells. This result partially explains the regenerative potential of autologous micro-SEFFI tissue grafts. In addition, using this novel Celector® technology, tissues used for aging treatment were characterized analytically, and the adipose tissue composition was evaluated with no need for extra sample processing.

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

PubMed

Shackney, Stanley; Emlet, David R; Pollice, Agnese; Smith, Charles; Brown, Kathryn; Kociban, Deborah

2006-01-01

Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. Copyright (c) 2005 Wiley-Liss, Inc.

A low-volume cavity ring-down spectrometer for sample-limited applications

NASA Astrophysics Data System (ADS)

Stowasser, C.; Farinas, A. D.; Ware, J.; Wistisen, D. W.; Rella, C.; Wahl, E.; Crosson, E.; Blunier, T.

2014-08-01

In atmospheric and environmental sciences, optical spectrometers are used for the measurements of greenhouse gas mole fractions and the isotopic composition of water vapor or greenhouse gases. The large sample cell volumes (tens of milliliters to several liters) in commercially available spectrometers constrain the usefulness of such instruments for applications that are limited in sample size and/or need to track fast variations in the sample stream. In an effort to make spectrometers more suitable for sample-limited applications, we developed a low-volume analyzer capable of measuring mole fractions of methane and carbon monoxide based on a commercial cavity ring-down spectrometer. The instrument has a small sample cell (9.6 ml) and can selectively be operated at a sample cell pressure of 140, 45, or 20 Torr (effective internal volume of 1.8, 0.57, and 0.25 ml). We present the new sample cell design and the flow path configuration, which are optimized for small sample sizes. To quantify the spectrometer's usefulness for sample-limited applications, we determine the renewal rate of sample molecules within the low-volume spectrometer. Furthermore, we show that the performance of the low-volume spectrometer matches the performance of the standard commercial analyzers by investigating linearity, precision, and instrumental drift.

Microfluidic, marker-free isolation of circulating tumor cells from blood samples

PubMed Central

Karabacak, Nezihi Murat; Spuhler, Philipp S; Fachin, Fabio; Lim, Eugene J; Pai, Vincent; Ozkumur, Emre; Martel, Joseph M; Kojic, Nikola; Smith, Kyle; Chen, Pin-i; Yang, Jennifer; Hwang, Henry; Morgan, Bailey; Trautwein, Julie; Barber, Thomas A; Stott, Shannon L; Maheswaran, Shyamala; Kapur, Ravi; Haber, Daniel A; Toner, Mehmet

2014-01-01

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen–independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2–5 d. PMID:24577360

Hyperbaric hydrothermal atomic force microscope

DOEpatents

Knauss, Kevin G.; Boro, Carl O.; Higgins, Steven R.; Eggleston, Carrick M.

2002-01-01

A hyperbaric hydrothermal atomic force microscope (AFM) is provided to image solid surfaces in fluids, either liquid or gas, at pressures greater than normal atmospheric pressure. The sample can be heated and its surface imaged in aqueous solution at temperatures greater than 100.degree. C. with less than 1 nm vertical resolution. A gas pressurized microscope base chamber houses the stepper motor and piezoelectric scanner. A chemically inert, flexible membrane separates this base chamber from the sample cell environment and constrains a high temperature, pressurized liquid or gas in the sample cell while allowing movement of the scanner. The sample cell is designed for continuous flow of liquid or gas through the sample environment.

Hyperbaric Hydrothermal Atomic Force Microscope

DOEpatents

Knauss, Kevin G.; Boro, Carl O.; Higgins, Steven R.; Eggleston, Carrick M.

2003-07-01

A hyperbaric hydrothermal atomic force microscope (AFM) is provided to image solid surfaces in fluids, either liquid or gas, at pressures greater than normal atmospheric pressure. The sample can be heated and its surface imaged in aqueous solution at temperatures greater than 100.degree. C. with less than 1 nm vertical resolution. A gas pressurized microscope base chamber houses the stepper motor and piezoelectric scanner. A chemically inert, flexible membrane separates this base chamber from the sample cell environment and constrains a high temperature, pressurized liquid or gas in the sample cell while allowing movement of the scanner. The sample cell is designed for continuous flow of liquid or gas through the sample environment.

Enrichment and Detection of Escherichia coli O157:H7 from Water Samples Using an Antibody Modified Microfluidic Chip

PubMed Central

Dharmasiri, Udara; Witek, Małgorzata A.; Adams, Andre A.; Osiri, John K.; Hupert, Mateusz L.; Bianchi, Thomas S.; Roelke, Daniel L.; Soper, Steven A.

2010-01-01

Low abundant (<100 cells mL-1) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip. The poly(methylmethacrylate), PMMA, chip contained 8 devices each equipped with 16 curvilinear high aspect ratio channels that were covalently decorated with polyclonal anti-O157 antibodies (pAb) and could search for rare cells through a pAb mediated process. The chip could process independently 8 different samples or one sample using 8 different parallel inputs to increase volume processing throughput. After cell enrichment, cells were released and enumerated using bench top real-time quantitative PCR, targeting genes which effectively discriminated the O157:H7 serotype from other non-pathogenic bacteria. The recovery of target cells from water samples was determined to be ~72%, and the limit-of-detection was found to be 6 colony forming units (cfu) using the slt1 gene as a reporter. We subsequently performed analysis of lake and waste water samples. The simplicity in manufacturing and ease of operation makes this device attractive for the selection of pathogenic species from a variety of water supplies suspected of containing bacterial pathogens at extremely low frequencies. PMID:20218574

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

PubMed

Sediq, A S; Klem, R; Nejadnik, M R; Meij, P; Jiskoot, Wim

2018-05-30

To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.

Biocompatible, smooth, plasma-treated nickel-titanium surface--an adequate platform for cell growth.

PubMed

Chrzanowski, W; Szade, J; Hart, A D; Knowles, J C; Dalby, M J

2012-02-01

High nickel content is believed to reduce the number of biomedical applications of nickel-titanium alloy due to the reported toxicity of nickel. The reduction in nickel release and minimized exposure of the cell to nickel can optimize the biocompatibility of the alloy and increase its use in the application where its shape memory effects and pseudoelasticity are particularly useful, e.g., spinal implants. Many treatments have been tried to improve the biocompatibility of Ni-Ti, and results suggest that a native, smooth surface could provide sufficient tolerance, biologically. We hypothesized that the native surface of nickel-titanium supports cell differentiation and insures good biocompatibility. Three types of surface modifications were investigated: thermal oxidation, alkali treatment, and plasma sputtering, and compared with smooth, ground surface. Thermal oxidation caused a drop in surface nickel content, while negligible chemistry changes were observed for plasma-modified samples when compared with control ground samples. In contrast, alkali treatment caused significant increase in surface nickel concentration and accelerated nickel release. Nickel release was also accelerated in thermally oxidized samples at 600 °C, while in other samples it remained at low level. Both thermal oxidation and alkali treatment increased the roughness of the surface, but mean roughness R(a) was significantly greater for the alkali-treated ones. Ground and plasma-modified samples had 'smooth' surfaces with R(a)=4 nm. Deformability tests showed that the adhesion of the surface layers on samples oxidized at 600 °C and alkali treatment samples was not sufficient; the layer delaminated upon deformation. It was observed that the cell cytoskeletons on the samples with a high nickel content or release were less developed, suggesting some negative effects of nickel on cell growth. These effects were observed primarily during initial cell contact with the surface. The most favorable cell responses were observed for ground and plasma-sputtered surfaces. These studies indicated that smooth, plasma-modified surfaces provide sufficient properties for cells to grow. © The Author(s), 2011.

Cytometer on a chip

NASA Technical Reports Server (NTRS)

Lynes, Michael A. (Inventor); Fernandez, Salvador M. (Inventor)

2010-01-01

An assay technique for label-free, highly parallel, qualitative and quantitative detection of specific cell populations in a sample and for assessing cell functional status, cell-cell interactions and cellular responses to drugs, environmental toxins, bacteria, viruses and other factors that may affect cell function. The technique includes a) creating a first array of binding regions in a predetermined spatial pattern on a sensor surface capable of specifically binding the cells to be assayed; b) creating a second set of binding regions in specific spatial patterns relative to the first set designed to efficiently capture potential secreted or released products from cells captured on the first set of binding regions; c) contacting the sensor surface with the sample, and d) simultaneously monitoring the optical properties of all the binding regions of the sensor surface to determine the presence and concentration of specific cell populations in the sample and their functional status by detecting released or secreted bioproducts.

Inhalation Frequency Controls Reformatting of Mitral/Tufted Cell Odor Representations in the Olfactory Bulb.

PubMed

Díaz-Quesada, Marta; Youngstrom, Isaac A; Tsuno, Yusuke; Hansen, Kyle R; Economo, Michael N; Wachowiak, Matt

2018-02-28

In mammals, olfactory sensation depends on inhalation, which controls activation of sensory neurons and temporal patterning of central activity. Odor representations by mitral and tufted (MT) cells, the main output from the olfactory bulb (OB), reflect sensory input as well as excitation and inhibition from OB circuits, which may change as sniff frequency increases. To test the impact of sampling frequency on MT cell odor responses, we obtained whole-cell recordings from MT cells in anesthetized male and female mice while varying inhalation frequency via tracheotomy, allowing comparison of inhalation-linked responses across cells. We characterized frequency effects on MT cell responses during inhalation of air and odorants using inhalation pulses and also "playback" of sniffing recorded from awake mice. Inhalation-linked changes in membrane potential were well predicted across frequency from linear convolution of 1 Hz responses; and, as frequency increased, near-identical temporal responses could emerge from depolarizing, hyperpolarizing, or multiphasic MT responses. However, net excitation was not well predicted from 1 Hz responses and varied substantially across MT cells, with some cells increasing and others decreasing in spike rate. As a result, sustained odorant sampling at higher frequencies led to increasing decorrelation of the MT cell population response pattern over time. Bulk activation of sensory inputs by optogenetic stimulation affected MT cells more uniformly across frequency, suggesting that frequency-dependent decorrelation emerges from odor-specific patterns of activity in the OB network. These results suggest that sampling behavior alone can reformat early sensory representations, possibly to optimize sensory perception during repeated sampling. SIGNIFICANCE STATEMENT Olfactory sensation in mammals depends on inhalation, which increases in frequency during active sampling of olfactory stimuli. We asked how inhalation frequency can shape the neural coding of odor information by recording from projection neurons of the olfactory bulb while artificially varying odor sampling frequency in the anesthetized mouse. We found that sampling an odor at higher frequencies led to diverse changes in net responsiveness, as measured by action potential output, that were not predicted from low-frequency responses. These changes led to a reorganization of the pattern of neural activity evoked by a given odorant that occurred preferentially during sustained, high-frequency inhalation. These results point to a novel mechanism for modulating early sensory representations solely as a function of sampling behavior. Copyright © 2018 the authors 0270-6474/18/382189-18$15.00/0.

Cell purification: a new challenge for biobanks.

PubMed

Almeida, Maria; García-Montero, Andres C; Orfao, Alberto

2014-01-01

Performing '-omics' analyses on heterogeneous biological tissue samples, such as blood or bone marrow, can lead to biased or even erroneous results, particularly when the targeted cells and/or molecules are present at relatively low percentages/amounts. In such cases, whole sample analysis will most probably dilute and mask the features of the cell and/or molecules of interest, and this will negatively impact the results and their interpretation. Therefore, frequently it is critically important to have well-characterized and high-quality purified cell populations for the reliable detection of subtle variations in their specific features, such as gene expression profile, protein expression pattern and metabolic status. Biobanks are technological platforms which aim to provide researchers access to a large number of high-quality biological samples and their associated data, particularly to support high-quality scientific and clinical research projects, and such projects will benefit enormously by having access to high-quality purified cell populations or their biological components (e.g. DNA, RNA, proteins). Therefore, a clear opportunity exists for preparative cell sorting techniques in biobanks. Although multiple different cell purification approaches exist or are under development (e.g. cell purification techniques based on cell adherence, density and/or cell size properties, methods based on antibody binding as well as new lab-on-a-chip purification techniques), the choice for a specific technology depends on multiple variables, including cell recovery, purity and yield, among others. In addition, most cell purification approaches are not well suited for high-throughput (HT) purification of multiple cell populations coexisting in a sample. Here we review the most (currently) used cell sorting methods that may be applied for sample preparation in biobanks. For the different approaches, technical considerations about their advantages and limitations are highlighted, and the requirements to be met by a HT cell sorting technology to be used in biobanks are also discussed.

Automated cell counts on CSF samples: A multicenter performance evaluation of the GloCyte system.

PubMed

Hod, E A; Brugnara, C; Pilichowska, M; Sandhaus, L M; Luu, H S; Forest, S K; Netterwald, J C; Reynafarje, G M; Kratz, A

2018-02-01

Automated cell counters have replaced manual enumeration of cells in blood and most body fluids. However, due to the unreliability of automated methods at very low cell counts, most laboratories continue to perform labor-intensive manual counts on many or all cerebrospinal fluid (CSF) samples. This multicenter clinical trial investigated if the GloCyte System (Advanced Instruments, Norwood, MA), a recently FDA-approved automated cell counter, which concentrates and enumerates red blood cells (RBCs) and total nucleated cells (TNCs), is sufficiently accurate and precise at very low cell counts to replace all manual CSF counts. The GloCyte System concentrates CSF and stains RBCs with fluorochrome-labeled antibodies and TNCs with nucleic acid dyes. RBCs and TNCs are then counted by digital image analysis. Residual adult and pediatric CSF samples obtained for clinical analysis at five different medical centers were used for the study. Cell counts were performed by the manual hemocytometer method and with the GloCyte System following the same protocol at all sites. The limits of the blank, detection, and quantitation, as well as precision and accuracy of the GloCyte, were determined. The GloCyte detected as few as 1 TNC/μL and 1 RBC/μL, and reliably counted as low as 3 TNCs/μL and 2 RBCs/μL. The total coefficient of variation was less than 20%. Comparison with cell counts obtained with a hemocytometer showed good correlation (>97%) between the GloCyte and the hemocytometer, including at very low cell counts. The GloCyte instrument is a precise, accurate, and stable system to obtain red cell and nucleated cell counts in CSF samples. It allows for the automated enumeration of even very low cell numbers, which is crucial for CSF analysis. These results suggest that GloCyte is an acceptable alternative to the manual method for all CSF samples, including those with normal cell counts. © 2017 John Wiley & Sons Ltd.

[Arf6, RalA and BIRC5 protein expression in non small cell lung cancer].

PubMed

Knizhnik, A V; Kovaleva, O B; Laktionov, K K; Mochal'nikova, V V; Komel'kov, A V; Chevkina, E M; Zborovskaia, I B

2011-01-01

Evaluation of tumor markers expression pattern which determines individual progression parameters is one of the major topics in molecular oncopathology research. This work presents research on expression analysis of several Ras-Ral associated signal transduction pathway proteins (Arf6, RalA and BIRC5) in accordance with clinical criteria in non small cell lung cancer patients. Using Western-blot analysis and RT-PCR Arf6, RalA and BIRC5 expression has been analyzed in parallel in 53 non small cell lung cancer samples of different origin. Arf6 protein expression was elevated in 55% non small cell lung cancer tumor samples in comparison with normal tissue. In the group of squamous cell lung cancer Arf6 expression elevation was observed more often. RalA protein expression was decreased in comparison to normal tissue samples in 64% of non small cell lung cancer regardless to morphological structure. Correlation between RalA protein expression decrease and absence of regional metastases was revealed for squamous cell lung cancer. BIRC5 protein expression in tumor samples versus corresponding normal tissue was 1.3 times more often elevated in the squamous cell lung cancer group (in 76% tumor samples). At the same time elevation of BIRC5 expression was fixed only in 63% of adenocarcinoma tumor samples. A statistically significant decrease (p = 0.0158) of RalA protein expression and increase (p = 0.0498) of Arf6 protein expression in comparison with normal tissue was found for T1-2N0M0 and T1-2N1-2M0 groups of squamous cell lung cancer correspondingly.

Identification of immune cell infiltration in hematoxylin-eosin stained breast cancer samples: texture-based classification of tissue morphologies

NASA Astrophysics Data System (ADS)

Turkki, Riku; Linder, Nina; Kovanen, Panu E.; Pellinen, Teijo; Lundin, Johan

2016-03-01

The characteristics of immune cells in the tumor microenvironment of breast cancer capture clinically important information. Despite the heterogeneity of tumor-infiltrating immune cells, it has been shown that the degree of infiltration assessed by visual evaluation of hematoxylin-eosin (H and E) stained samples has prognostic and possibly predictive value. However, quantification of the infiltration in H and E-stained tissue samples is currently dependent on visual scoring by an expert. Computer vision enables automated characterization of the components of the tumor microenvironment, and texture-based methods have successfully been used to discriminate between different tissue morphologies and cell phenotypes. In this study, we evaluate whether local binary pattern texture features with superpixel segmentation and classification with support vector machine can be utilized to identify immune cell infiltration in H and E-stained breast cancer samples. Guided with the pan-leukocyte CD45 marker, we annotated training and test sets from 20 primary breast cancer samples. In the training set of arbitrary sized image regions (n=1,116) a 3-fold cross-validation resulted in 98% accuracy and an area under the receiver-operating characteristic curve (AUC) of 0.98 to discriminate between immune cell -rich and - poor areas. In the test set (n=204), we achieved an accuracy of 96% and AUC of 0.99 to label cropped tissue regions correctly into immune cell -rich and -poor categories. The obtained results demonstrate strong discrimination between immune cell -rich and -poor tissue morphologies. The proposed method can provide a quantitative measurement of the degree of immune cell infiltration and applied to digitally scanned H and E-stained breast cancer samples for diagnostic purposes.

Determination of Cu Environments in the Cyanobacterium Anabaena flos-aquae by X-Ray Absorption Spectroscopy

PubMed Central

Kretschmer, X. C.; Meitzner, G.; Gardea-Torresdey, J. L.; Webb, R.

2004-01-01

Whole cells and peptidoglycan isolated from cell walls of the cyanobacterium Anabaena flos-aquae were lyophilized and used at pH 2 and pH 5 in Cu(II) binding studies. X-ray absorption spectra measured at the Cu K-edge were used to determine the oxidation states and chemical environments of Cu species in the whole-cell and peptidoglycan samples. In the whole-cell samples, most of the Cu retained at both pH values was coordinated by phosphate ligands. The whole-cell fractions contained significant concentrations of Cu(I) as well as Cu(II). An X-ray absorption near-edge spectrum analysis suggested that Cu(I) was coordinated by amine and thiol ligands. An analysis of the peptidoglycan fractions found that more Cu was adsorbed by the peptidoglycan fraction prepared at pH 5, due to increased chelation by amine and carboxyl ligands. The peptidoglycan fractions, also referred to as the cell wall fractions, contained little or no Cu(I). The Cu loading level was 30 times higher in the cell wall sample prepared at pH 5 than in the sample prepared at pH 2. Amine and bidentate carboxyl ligands had similar relative levels of importance in cell wall peptidoglycan samples prepared at both pH values, but phosphate coordination was insignificant. PMID:14766554

A new apparatus design for high temperature (up to 950 °C) quasi-elastic neutron scattering in a controlled gaseous environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al-Wahish, Amal; Armitage, D.; Hill, B.

A design for a sample cell system suitable for high temperature Quasi-Elastic Neutron Scattering (QENS) experiments is presented. The apparatus was developed at the Spallation Neutron Source in Oak Ridge National Lab where it is currently in use. The design provides a special sample cell environment under controlled humid or dry gas flow over a wide range of temperature up to 950 °C. Using such a cell, chemical, dynamical, and physical changes can be studied in situ under various operating conditions. While the cell combined with portable automated gas environment system is especially useful for in situ studies of microscopic dynamicsmore » under operational conditions that are similar to those of solid oxide fuel cells, it can additionally be used to study a wide variety of materials, such as high temperature proton conductors. The cell can also be used in many different neutron experiments when a suitable sample holder material is selected. The sample cell system has recently been used to reveal fast dynamic processes in quasi-elastic neutron scattering experiments, which standard probes (such as electrochemical impedance spectroscopy) could not detect. In this work, we outline the design of the sample cell system and present results demonstrating its abilities in high temperature QENS experiments.« less

A new apparatus design for high temperature (up to 950°C) quasi-elastic neutron scattering in a controlled gaseous environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

al-Wahish, Amal; Armitage, D.; al-Binni, U.

Our design for a sample cell system suitable for high temperature Quasi-Elastic Neutron Scattering (QENS) experiments is presented. The apparatus was developed at the Spallation Neutron Source in Oak Ridge National Lab where it is currently in use. The design provides a special sample cell environment under controlled humid or dry gas flow over a wide range of temperature up to 950°C. Using such a cell, chemical, dynamical, and physical changes can be studied in situ under various operating conditions. And while the cell combined with portable automated gas environment system is especially useful for in situ studies of microscopicmore » dynamics under operational conditions that are similar to those of solid oxide fuel cells, it can additionally be used to study a wide variety of materials, such as high temperature protonconductors. The cell can also be used in many different neutron experiments when a suitable sample holder material is selected. Finally, the sample cell system has recently been used to reveal fast dynamic processes in quasi-elastic neutron scattering experiments, which standard probes (such as electrochemical impedance spectroscopy) could not detect. In this work, we outline the design of the sample cell system and present results demonstrating its abilities in high temperature QENS experiments.« less

Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

NASA Astrophysics Data System (ADS)

Chandra, Subhash

2008-12-01

Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2+) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells provided a rule-of-thumb criterion for the validation of sample preparation. The fractured freeze-dried cells allowed 3-D SIMS imaging and localization of 13C 15N labeled molecules and therapeutic drugs containing an elemental tag. Examples are shown to demonstrate that both diffusible elements and molecules are prone to artifact-induced relocation at subcellular scale if the sample preparation is compromised. The sample preparation is problem dependent and may vary widely between the diverse sample types of biological systems and the type of instrument used for SIMS analysis. The sample preparation, however, must be validated so that SIMS can be applied with confidence in biology and medicine.

Wide-field fluorescent microscopy on a cell-phone.

PubMed

Zhu, Hongying; Yaglidere, Oguzhan; Su, Ting-Wei; Tseng, Derek; Ozcan, Aydogan

2011-01-01

We demonstrate wide-field fluorescent imaging on a cell-phone, using compact and cost-effective optical components that are mechanically attached to the existing camera unit of the cell-phone. Battery powered light-emitting diodes (LEDs) are used to side-pump the sample of interest using butt-coupling. The pump light is guided within the sample cuvette to excite the specimen uniformly. The fluorescent emission from the sample is then imaged with an additional lens that is put in front of the existing lens of the cell-phone camera. Because the excitation occurs through guided waves that propagate perpendicular to the detection path, an inexpensive plastic color filter is sufficient to create the dark-field background needed for fluorescent imaging. The imaging performance of this light-weight platform (~28 grams) is characterized with red and green fluorescent microbeads, achieving an imaging field-of-view of ~81 mm(2) and a spatial resolution of ~10 μm, which is enhanced through digital processing of the captured cell-phone images using compressive sampling based sparse signal recovery. We demonstrate the performance of this cell-phone fluorescent microscope by imaging labeled white-blood cells separated from whole blood samples as well as water-borne pathogenic protozoan parasites such as Giardia Lamblia cysts.

Automated image analysis for quantitative fluorescence in situ hybridization with environmental samples.

PubMed

Zhou, Zhi; Pons, Marie Noëlle; Raskin, Lutgarde; Zilles, Julie L

2007-05-01

When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An automated image analysis program that detects cells from 4',6'-diamidino-2-phenylindole (DAPI) micrographs and extracts the maximum and mean fluorescence intensities for each cell from corresponding FISH images was developed with the software Visilog. Intensity thresholds were not consistent even for duplicate analyses, so alternative ways of classifying signals were investigated. In the resulting method, the intensity data were divided into clusters using fuzzy c-means clustering, and the resulting clusters were classified as target (positive) or nontarget (negative). A manual quality control confirmed this classification. With this method, 50.4, 72.1, and 64.9% of the cells in two swine manure samples and one soil sample, respectively, were positive as determined with a 16S rRNA-targeted bacterial probe (S-D-Bact-0338-a-A-18). Manual counting resulted in corresponding values of 52.3, 70.6, and 61.5%, respectively. In two swine manure samples and one soil sample 21.6, 12.3, and 2.5% of the cells were positive with an archaeal probe (S-D-Arch-0915-a-A-20), respectively. Manual counting resulted in corresponding values of 22.4, 14.0, and 2.9%, respectively. This automated method should facilitate quantitative analysis of FISH images for a variety of complex environmental samples.

Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies.

PubMed

Reynolds, K A; Roll, K; Fujioka, R S; Gerba, C P; Pepper, I L

1998-06-01

The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase-polymerase chain reaction (RT-PCR). Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.

MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples

EPA Science Inventory

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from ...

Design and performance of tapered cubic anvil used for achieving higher pressure and larger sample cell

NASA Astrophysics Data System (ADS)

Han, Qi-Gang; Yang, Wen-Ke; Zhu, Pin-Wen; Ban, Qing-Chu; Yan, Ni; Zhang, Qiang

2013-07-01

In order to increase the maximum cell pressure of the cubic high pressure apparatus, we have developed a new structure of tungsten carbide cubic anvil (tapered cubic anvil), based on the principle of massive support and lateral support. Our results indicated that the tapered cubic anvil has some advantages. First, tapered cubic anvil can push the transfer rate of pressure well into the range above 36.37% compare to the conventional anvil. Second, the rate of failure crack decreases about 11.20% after the modification of the conventional anvil. Third, the limit of static high-pressure in the sample cell can be extended to 13 GPa, which can increase the maximum cell pressure about 73.3% than that of the conventional anvil. Fourth, the volume of sample cell compressed by tapered cubic anvils can be achieved to 14.13 mm3 (3 mm diameter × 2 mm long), which is three and six orders of magnitude larger than that of double-stage apparatus and diamond anvil cell, respectively. This work represents a relatively simple method for achieving higher pressures and larger sample cell.

Comparative Assessment of a Self-sampling Device and Gynecologist Sampling for Cytology and HPV DNA Detection in a Rural and Low Resource Setting: Malaysian Experience.

PubMed

Latiff, Latiffah A; Ibrahim, Zaidah; Pei, Chong Pei; Rahman, Sabariah Abdul; Akhtari-Zavare, Mehrnoosh

2015-01-01

This study was conducted to assess the agreement and differences between cervical self-sampling with a Kato device (KSSD) and gynecologist sampling for Pap cytology and human papillomavirus DNA (HPV DNA) detection. Women underwent self-sampling followed by gynecologist sampling during screening at two primary health clinics. Pap cytology of cervical specimens was evaluated for specimen adequacy, presence of endocervical cells or transformation zone cells and cytological interpretation for cells abnormalities. Cervical specimens were also extracted and tested for HPV DNA detection. Positive HPV smears underwent gene sequencing and HPV genotyping by referring to the online NCBI gene bank. Results were compared between samplings by Kappa agreement and McNemar test. For Pap specimen adequacy, KSSD showed 100% agreement with gynecologist sampling but had only 32.3% agreement for presence of endocervical cells. Both sampling showed 100% agreement with only 1 case detected HSIL favouring CIN2 for cytology result. HPV DNA detection showed 86.2%agreement (K=0.64, 95% CI 0.524-0.756, p=0.001) between samplings. KSSD and gynaecologist sampling identified high risk HPV in 17.3% and 23.9% respectively (p= 0.014). The self-sampling using Kato device can serve as a tool in Pap cytology and HPV DNA detection in low resource settings in Malaysia. Self-sampling devices such as KSSD can be used as an alternative technique to gynaecologist sampling for cervical cancer screening among rural populations in Malaysia.

Nucleolar structure and proliferation activity of Arabidopsis root cells from seedlings germinated on the International Space Station

NASA Astrophysics Data System (ADS)

Matía, Isabel; González-Camacho, Fernando; Marco, Roberto; Kiss, John Z.; Gasset, Gilbert; Medina, Francisco-Javier

Seeds of Arabidopsis thaliana were sent to the International Space Station in the "Cervantes Mission" (Spanish Soyuz Mission). Seed germination was initiated in flight by supplying culture medium. Seedlings were grown for 4 days at 22 °C, and growth was stopped by the addition of paraformaldehyde fixative. Once back on the ground, samples were immediately processed for microscopy. A ground control experiment was simultaneously replicated. Glutaraldehyde-fixed root cells from seedlings grown in the Biorack on board of the Space Shuttle (STS-84 Mission) in similar conditions were also ultrastructurally examined. The length of seedlings grown at 1 g was conspicuously shorter than parallel samples grown under microgravity. We examined the morphology of the root meristematic cells, with a focus on their nucleoli in the cortex and stele. In general, root cortical cells proliferate at a higher rate and their nucleoli are more active than those of stele cells. While the stele showed longer cells with larger nucleoli in the flight samples, cortical cells from space-grown seedlings were shorter, more numerous and more densely packed than ground controls. However, nucleoli were smaller and less active in fast proliferating flight cells than in the ground controls. This reduced level of ribosome synthesis in the flight samples is probably the result of an accelerated cell cycle. An altered rate of cell proliferation may be detrimental for the plant and could be the reason for the reported smaller size of older space-grown seedlings. Finally, two-dimensional protein electrophoresis showed noticeable differences between space samples and ground controls.

Intact preservation of environmental samples by freezing under an alternating magnetic field.

PubMed

Morono, Yuki; Terada, Takeshi; Yamamoto, Yuhji; Xiao, Nan; Hirose, Takehiro; Sugeno, Masaya; Ohwada, Norio; Inagaki, Fumio

2015-04-01

The study of environmental samples requires a preservation system that stabilizes the sample structure, including cells and biomolecules. To address this fundamental issue, we tested the cell alive system (CAS)-freezing technique for subseafloor sediment core samples. In the CAS-freezing technique, an alternating magnetic field is applied during the freezing process to produce vibration of water molecules and achieve a stable, super-cooled liquid phase. Upon further cooling, the temperature decreases further, achieving a uniform freezing of sample with minimal ice crystal formation. In this study, samples were preserved using the CAS and conventional freezing techniques at 4, -20, -80 and -196 (liquid nitrogen) °C. After 6 months of storage, microbial cell counts by conventional freezing significantly decreased (down to 10.7% of initial), whereas that by CAS-freezing resulted in minimal. When Escherichia coli cells were tested under the same freezing conditions and storage for 2.5 months, CAS-frozen E. coli cells showed higher viability than the other conditions. In addition, an alternating magnetic field does not impact on the direction of remanent magnetization in sediment core samples, although slight partial demagnetization in intensity due to freezing was observed. Consequently, our data indicate that the CAS technique is highly useful for the preservation of environmental samples. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Axial tomography in 3D live cell microscopy

NASA Astrophysics Data System (ADS)

Richter, Verena; Bruns, Sarah; Bruns, Thomas; Piper, Mathis; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

2017-07-01

A miniaturized setup for sample rotation on a microscope stage has been developed, combined with light sheet, confocal or structured illumination microscopy and applied to living cells as well as to small organisms. This setup permits axial tomography with improved visualization of single cells or small cell clusters as well as an enhanced effective 3D resolution upon sample rotation.

Growing cell-phone population and noncoverage bias in traditional random digit dial telephone health surveys.

PubMed

Lee, Sunghee; Brick, J Michael; Brown, E Richard; Grant, David

2010-08-01

Examine the effect of including cell-phone numbers in a traditional landline random digit dial (RDD) telephone survey. The 2007 California Health Interview Survey (CHIS). CHIS 2007 is an RDD telephone survey supplementing a landline sample in California with a sample of cell-only (CO) adults. We examined the degree of bias due to exclusion of CO populations and compared a series of demographic and health-related characteristics by telephone usage. When adjusted for noncoverage in the landline sample through weighting, the potential noncoverage bias due to excluding CO adults in landline telephone surveys is diminished. Both CO adults and adults who have both landline and cell phones but mostly use cell phones appear different from other telephone usage groups. Controlling for demographic differences did not attenuate the significant distinctiveness of cell-mostly adults. While careful weighting can mitigate noncoverage bias in landline telephone surveys, the rapid growth of cell-phone population and their distinctive characteristics suggest it is important to include a cell-phone sample. Moreover, the threat of noncoverage bias in telephone health survey estimates could mislead policy makers with possibly serious consequences for their ability to address important health policy issues.

Dielectrophoresis-Based Sample Handling in General-Purpose Programmable Diagnostic Instruments

PubMed Central

Gascoyne, Peter R. C.; Vykoukal, Jody V.

2009-01-01

As the molecular origins of disease are better understood, the need for affordable, rapid, and automated technologies that enable microscale molecular diagnostics has become apparent. Widespread use of microsystems that perform sample preparation and molecular analysis could ensure that the benefits of new biomedical discoveries are realized by a maximum number of people, even those in environments lacking any infrastructure. While progress has been made in developing miniaturized diagnostic systems, samples are generally processed off-device using labor-intensive and time-consuming traditional sample preparation methods. We present the concept of an integrated programmable general-purpose sample analysis processor (GSAP) architecture where raw samples are routed to separation and analysis functional blocks contained within a single device. Several dielectrophoresis-based methods that could serve as the foundation for building GSAP functional blocks are reviewed including methods for cell and particle sorting, cell focusing, cell ac impedance analysis, cell lysis, and the manipulation of molecules and reagent droplets. PMID:19684877

A novel sample preparation method to avoid influence of embedding medium during nano-indentation

NASA Astrophysics Data System (ADS)

Meng, Yujie; Wang, Siqun; Cai, Zhiyong; Young, Timothy M.; Du, Guanben; Li, Yanjun

2013-02-01

The effect of the embedding medium on the nano-indentation measurements of lignocellulosic materials was investigated experimentally using nano-indentation. Both the reduced elastic modulus and the hardness of non-embedded cell walls were found to be lower than those of the embedded samples, proving that the embedding medium used for specimen preparation on cellulosic material during nano-indentation can modify cell-wall properties. This leads to structural and chemical changes in the cell-wall constituents, changes that may significantly alter the material properties. Further investigation was carried out to detect the influence of different vacuum times on the cell-wall mechanical properties during the embedding procedure. Interpretation of the statistical analysis revealed no linear relationships between vacuum time and the mechanical properties of cell walls. The quantitative measurements confirm that low-viscosity resin has a rapid penetration rate early in the curing process. Finally, a novel sample preparation method aimed at preventing resin diffusion into lignocellulosic cell walls was developed using a plastic film to wrap the sample before embedding. This method proved to be accessible and straightforward for many kinds of lignocellulosic material, but is especially suitable for small, soft samples.

Cellular network entropy as the energy potential in Waddington's differentiation landscape

PubMed Central

Banerji, Christopher R. S.; Miranda-Saavedra, Diego; Severini, Simone; Widschwendter, Martin; Enver, Tariq; Zhou, Joseph X.; Teschendorff, Andrew E.

2013-01-01

Differentiation is a key cellular process in normal tissue development that is significantly altered in cancer. Although molecular signatures characterising pluripotency and multipotency exist, there is, as yet, no single quantitative mark of a cellular sample's position in the global differentiation hierarchy. Here we adopt a systems view and consider the sample's network entropy, a measure of signaling pathway promiscuity, computable from a sample's genome-wide expression profile. We demonstrate that network entropy provides a quantitative, in-silico, readout of the average undifferentiated state of the profiled cells, recapitulating the known hierarchy of pluripotent, multipotent and differentiated cell types. Network entropy further exhibits dynamic changes in time course differentiation data, and in line with a sample's differentiation stage. In disease, network entropy predicts a higher level of cellular plasticity in cancer stem cell populations compared to ordinary cancer cells. Importantly, network entropy also allows identification of key differentiation pathways. Our results are consistent with the view that pluripotency is a statistical property defined at the cellular population level, correlating with intra-sample heterogeneity, and driven by the degree of signaling promiscuity in cells. In summary, network entropy provides a quantitative measure of a cell's undifferentiated state, defining its elevation in Waddington's landscape. PMID:24154593

Prevalence, isolation and characterization of Bacillus cereus strains from rice of local cultivators of Sabah, Sarawak, and Peninsular Malaysia

NASA Astrophysics Data System (ADS)

Sawei, Jelin; Sani, Norrakiah Abdullah

2016-11-01

Bacillus cereus is a spore-forming, facultative anaerobic, motile microorganism that has been identified as a causative agent of two types of gastrointestinal diseases such as emetic and diarrhea. This foodborne pathogen is found in both vegetative cells and endospores form in foods such as rice either raw or cooked. The aim of this study is to investigate and determine the prevalence, characterize and identify the isolation of vegetative cells and endospores of B. cereus in thirty varieties (n=3) of raw rice from Sabah, Sarawak and Peninsular Malaysia. A total of 90 (n=90) raw rice were examined and 84 (93.33%) samples were positive to vegetative cells of B. cereus. However, only 32 (35.56%) samples were positive for endospore cells that able to germinate after samples were heated at 75°C for 15 mins. The mean log cfu/g for vegetative cells were higher range (0.00 - 4.1533) than visible endospores (0.00 - 3.7533 mean log cfu/g). Sample of raw red rice (UKMRC9) had significantly higher contamination by both vegetative cells and endospores at p<0.05, than the other raw rice samples.

Application of imaging spectrometer in gas analysis by Raman scattering

NASA Astrophysics Data System (ADS)

Zuo, Duluo; Yu, Anlan; Li, Zhe; Wang, Xingbing; Xiong, Youhui

2015-09-01

Spontaneous Raman scattering is an effective technique in gas analysis, but the detection of minor constituents is difficult because of the low signal level and the usually existed background. Imaging spectrometer can provide highly spatial resolved spectra, so it should be much easier to pick up Raman signal of minor constituents from the Raman/fluorescence background of the sample cell and transporting optics compared with the widely used fiber-coupled spectrometers. For this reason, an imaging spectrometer was constructed from transmitting volume phase holographic grating, camera lenses and CCD detector. When it was used to analyze the gas sample in metal-lined capillary, which is a sample cell believed with great enhancement of Raman signal, the background was compressed obviously. When it was used to analyze the gas in a sample cell including a parabolic reflector, only weak background signal was observed, as the wide separation between the collecting zone (the focus point of the parabolic surface) and the wall of sample cell benefitted to the analysis by imaging spectrometer. By using the last sample cell, the signal from CO2 in ambient air was able to be found by an exposure time about 20 sec, and limits of detection for H2, CO2 and CO were estimated as 60 ppm, 100 ppm and 300 ppm respectively by the results of a longer exposure time. These results show that an imaging spectrometer paired with a well-arranged sample cell will lower the detecting limit effectively.

RNA-Stabilized Whole Blood Samples but Not Peripheral Blood Mononuclear Cells Can Be Stored for Prolonged Time Periods Prior to Transcriptome Analysis

PubMed Central

Debey-Pascher, Svenja; Hofmann, Andrea; Kreusch, Fatima; Schuler, Gerold; Schuler-Thurner, Beatrice; Schultze, Joachim L.; Staratschek-Jox, Andrea

2011-01-01

Microarray-based transcriptome analysis of peripheral blood as surrogate tissue has become an important approach in clinical implementations. However, application of gene expression profiling in routine clinical settings requires careful consideration of the influence of sample handling and RNA isolation methods on gene expression profile outcome. We evaluated the effect of different sample preservation strategies (eg, cryopreservation of peripheral blood mononuclear cells or freezing of PAXgene-stabilized whole blood samples) on gene expression profiles. Expression profiles obtained from cryopreserved peripheral blood mononuclear cells differed substantially from those of their nonfrozen counterpart samples. Furthermore, expression profiles in cryopreserved peripheral blood mononuclear cell samples were found to undergo significant alterations with increasing storage period, whereas long-term freezing of PAXgene RNA stabilized whole blood samples did not significantly affect stability of gene expression profiles. This report describes important technical aspects contributing toward the establishment of robust and reliable guidance for gene expression studies using peripheral blood and provides a promising strategy for reliable implementation in routine handling for diagnostic purposes. PMID:21704280

Supplying osteogenesis to dead bone using an osteogenic matrix cell sheet.

PubMed

Uchihara, Yoshinobu; Akahane, Manabu; Okuda, Akinori; Shimizu, Takamasa; Masuda, Keisuke; Kira, Tsutomu; Kawate, Kenji; Tanaka, Yasuhito

2018-02-22

To evaluate whether osteogenic matrix cell sheets can supply osteogenesis to dead bone. Femur bone fragments (5 mm in length) were obtained from Fisher 344 rats and irradiated by a single exposure of 60 Gy to produce bones that were no longer viable. Osteogenic matrix cell sheets were created from rat bone marrow-derived stromal cells (BMSCs). After wrapping the dead bone with an osteogenic matrix cell sheet, it was subcutaneously transplanted into the back of a rat and harvested after 4 weeks. Bone formation around the dead bone was evaluated by X-ray imaging and histology. Alkaline phosphatase (ALP) and osteocalcin (OC) mRNA expression levels were measured to confirm osteogenesis of the transplanted bone. The contribution of donor cells to bone formation was assessed using the Sry gene and PKH26. After the cell sheet was transplanted together with dead bone, X-ray images showed abundant calcification around the dead bone. In contrast, no newly formed bone was seen in samples that were transplanted without the cell sheet. Histological sections also showed newly formed bone around dead bone in samples transplanted with the cell sheet, whereas many empty lacunae and no newly formed bone were observed in samples transplanted without the cell sheet. ALP and OC mRNA expression levels were significantly higher in dead bones transplanted with cell sheets than in those without a cell sheet (P < 0.01). Sry gene expression and cells derived from cell sheets labeled with PKH26 were detected in samples transplanted with a cell sheet, indicating survival of donor cells after transplantation. Our study indicates that osteogenic matrix cell sheet transplantation can supply osteogenesis to dead bone. Copyright © 2018. Published by Elsevier B.V.

Automated cellular sample preparation using a Centrifuge-on-a-Chip.

PubMed

Mach, Albert J; Kim, Jae Hyun; Arshi, Armin; Hur, Soojung Claire; Di Carlo, Dino

2011-09-07

The standard centrifuge is a laboratory instrument widely used by biologists and medical technicians for preparing cell samples. Efforts to automate the operations of concentration, cell separation, and solution exchange that a centrifuge performs in a simpler and smaller platform have had limited success. Here, we present a microfluidic chip that replicates the functions of a centrifuge without moving parts or external forces. The device operates using a purely fluid dynamic phenomenon in which cells selectively enter and are maintained in microscale vortices. Continuous and sequential operation allows enrichment of cancer cells from spiked blood samples at the mL min(-1) scale, followed by fluorescent labeling of intra- and extra-cellular antigens on the cells without the need for manual pipetting and washing steps. A versatile centrifuge-analogue may open opportunities in automated, low-cost and high-throughput sample preparation as an alternative to the standard benchtop centrifuge in standardized clinical diagnostics or resource poor settings.

3D nanoscale imaging of the yeast, Schizosaccharomyces pombe, by full-field transmission X-ray microscopy at 5.4 keV.

PubMed

Chen, Jie; Yang, Yunhao; Zhang, Xiaobo; Andrews, Joy C; Pianetta, Piero; Guan, Yong; Liu, Gang; Xiong, Ying; Wu, Ziyu; Tian, Yangchao

2010-07-01

Three-dimensional (3D) nanoscale structures of the fission yeast, Schizosaccharomyces pombe, can be obtained by full-field transmission hard X-ray microscopy with 30 nm resolution using synchrotron radiation sources. Sample preparation is relatively simple and the samples are portable across various imaging environments, allowing for high-throughput sample screening. The yeast cells were fixed and double-stained with Reynold's lead citrate and uranyl acetate. We performed both absorption contrast and Zernike phase contrast imaging on these cells in order to test this method. The membranes, nucleus, and subcellular organelles of the cells were clearly visualized using absorption contrast mode. The X-ray images of the cells could be used to study the spatial distributions of the organelles in the cells. These results show unique structural information, demonstrating that hard X-ray microscopy is a complementary method for imaging and analyzing biological samples.

Metabolic Changes Caused by Ethinylestradiol in Human Breast Cancer Cells as Observed by NMR Spectroscopy

EPA Science Inventory

To conduct cell culture based metabolomics, we have developed a novel sample preparation method using adherent mammalian cells, which is rapid, effective, and exhibits greater metabolite retention by approximately a factor of 50 compared to the conventional sample preparation met...

Detection and capture of single circulating melanoma cells using photoacoustic flowmetry

NASA Astrophysics Data System (ADS)

O'Brien, Christine; Mosley, Jeffrey; Goldschmidt, Benjamin S.; Viator, John A.

2010-02-01

Photoacoustic flowmetry has been used to detect single circulating melanoma cells in vitro. Circulating melanoma cells are those cells that travel in the blood and lymph systems to create secondary tumors and are the hallmark of metastasis. This technique involves taking blood samples from patients, separating the white blood and melanoma cells from whole blood and irradiating them with a pulsed laser in a flowmetry set up. Rapid, visible wavelength laser pulses on the order of 5 ns can induce photoacoustic waves in melanoma cells due to their melanin content, while surrounding white blood cells remain acoustically passive. We have developed a system that identifies rare melanoma cells and captures them in 50 microliter volumes using suction applied near the photoacoustic detection chamber. The 50 microliter sample is then diluted and the experiment is repeated using the new sample until only a melanoma cell remains. We have tested this system on dyed microspheres ranging in size from 300 to 500 microns. Capture of circulating melanoma cells may provide the opportunity to study metastatic cells for basic understanding of the spread of cancer and to optimize patient specific therapies.

Elegant grapheme-phoneme correspondence: a periodic chart and singularity generalization unify decoding.

PubMed

Gates, Louis

2018-04-01

The accompanying article introduces highly transparent grapheme-phoneme relationships embodied within a Periodic table of decoding cells, which arguably presents the quintessential transparent decoding elements. The study then folds these cells into one highly transparent but simply stated singularity generalization-this generalization unifies the decoding cells (97% transparency). Deeper, the periodic table and singularity generalization together highlight the connectivity of the periodic cells. Moreover, these interrelated cells, coupled with the singularity generalization, clarify teaching targets and enable efficient learning of the letter-sound code. This singularity generalization, in turn, serves as a model for creating unified but easily stated subordinate generalizations for any one of the transparent cells or groups of cells shown within the tables. The article then expands the periodic cells into two tables of teacher-ready sample word lists-one table includes sample words for the basic and phonogram vowel cells, and the other table embraces word samples for the transparent consonant cells. The paper concludes with suggestions for teaching the cellular transparency embedded within reoccurring isolated words and running text to promote decoding automaticity of the periodic cells.

Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes.

PubMed

Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke; Saini, Sunil Kumar; Ramskov, Sofie; Donia, Marco; Such, Lina; Furness, Andrew J S; McGranahan, Nicholas; Rosenthal, Rachel; Straten, Per Thor; Szallasi, Zoltan; Svane, Inge Marie; Swanton, Charles; Quezada, Sergio A; Jakobsen, Søren Nyboe; Eklund, Aron Charles; Hadrup, Sine Reker

2016-10-01

Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10-100 distinct T-cell specificities in one sample. Here we use peptide-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.

White blood cell counting system

NASA Technical Reports Server (NTRS)

1972-01-01

The design, fabrication, and tests of a prototype white blood cell counting system for use in the Skylab IMSS are presented. The counting system consists of a sample collection subsystem, sample dilution and fluid containment subsystem, and a cell counter. Preliminary test results show the sample collection and the dilution subsystems are functional and fulfill design goals. Results for the fluid containment subsystem show the handling bags cause counting errors due to: (1) adsorption of cells to the walls of the container, and (2) inadequate cleaning of the plastic bag material before fabrication. It was recommended that another bag material be selected.

Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics

PubMed Central

Andersson, Ann-Catrin; Strömberg, Sara; Bäckvall, Helena; Kampf, Caroline; Uhlen, Mathias; Wester, Kenneth; Pontén, Fredrik

2006-01-01

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome. PMID:16957166

Detection and Characterization of Circulating Tumour Cells from Frozen Peripheral Blood Mononuclear Cells

PubMed Central

Lu, David; Graf, Ryon P.; Harvey, Melissa; Madan, Ravi A.; Heery, Christopher; Marte, Jennifer; Beasley, Sharon; Tsang, Kwong Y.; Krupa, Rachel; Louw, Jessica; Wahl, Justin; Bales, Natalee; Landers, Mark; Marrinucci, Dena; Schlom, Jeffrey; Gulley, James L.; Dittamore, Ryan

2015-01-01

Retrospective analysis of patient tumour samples is a cornerstone of clinical research. CTC biomarker characterization offers a non-invasive method to analyse patient samples. However, current CTC technologies require prospective blood collection, thereby reducing the ability to utilize archived clinical cohorts with long-term outcome data. We sought to investigate CTC recovery from frozen, archived patient PBMC pellets. Matched samples from both mCRPC patients and mock samples, which were prepared by spiking healthy donor blood with cultured prostate cancer cell line cells, were processed “fresh” via Epic CTC Platform or from “frozen” PBMC pellets. Samples were analysed for CTC enumeration and biomarker characterization via immunofluorescent (IF) biomarkers, fluorescence in-situ hybridization (FISH) and CTC morphology. In the frozen patient PMBC samples, the median CTC recovery was 18%, compared to the freshly processed blood. However, abundance and localization of cytokeratin (CK) and androgen receptor (AR) protein, as measured by IF, were largely concordant between the fresh and frozen CTCs. Furthermore, a FISH analysis of PTEN loss showed high concordance in fresh vs. frozen. The observed data indicate that CTC biomarker characterization from frozen archival samples is feasible and representative of prospectively collected samples. PMID:28936240

Scanning transmission ion microscopy mass measurements for quantitative trace element analysis within biological samples and validation using atomic force microscopy thickness measurements

NASA Astrophysics Data System (ADS)

Devès, Guillaume; Cohen-Bouhacina, Touria; Ortega, Richard

2004-10-01

We used the nuclear microprobe techniques, micro-PIXE (particle-induced X-ray emission), micro-RBS (Rutherford backscattering spectrometry) and scanning transmission ion microscopy (STIM) in order to perform the characterization of trace element content and spatial distribution within biological samples (dehydrated cultured cells, tissues). The normalization of PIXE results was usually expressed in terms of sample dry mass as determined by micro-RBS recorded simultaneously to micro-PIXE. However, the main limit of RBS mass measurement is the sample mass loss occurring during irradiation and which could be up to 30% of the initial sample mass. We present here a new methodology for PIXE normalization and quantitative analysis of trace element within biological samples based on dry mass measurement performed by mean of STIM. The validation of STIM cell mass measurements was obtained in comparison with AFM sample thickness measurements. Results indicated the reliability of STIM mass measurement performed on biological samples and suggested that STIM should be performed for PIXE normalization. Further information deriving from direct confrontation of AFM and STIM analysis could as well be obtained, like in situ measurements of cell specific gravity within cells compartment (nucleolus and cytoplasm).

Scanning electron microscopy coupled with energy-dispersive X-ray spectrometry for quick detection of sulfur-oxidizing bacteria in environmental water samples

NASA Astrophysics Data System (ADS)

Sun, Chengjun; Jiang, Fenghua; Gao, Wei; Li, Xiaoyun; Yu, Yanzhen; Yin, Xiaofei; Wang, Yong; Ding, Haibing

2017-01-01

Detection of sulfur-oxidizing bacteria has largely been dependent on targeted gene sequencing technology or traditional cell cultivation, which usually takes from days to months to carry out. This clearly does not meet the requirements of analysis for time-sensitive samples and/or complicated environmental samples. Since energy-dispersive X-ray spectrometry (EDS) can be used to simultaneously detect multiple elements in a sample, including sulfur, with minimal sample treatment, this technology was applied to detect sulfur-oxidizing bacteria using their high sulfur content within the cell. This article describes the application of scanning electron microscopy imaging coupled with EDS mapping for quick detection of sulfur oxidizers in contaminated environmental water samples, with minimal sample handling. Scanning electron microscopy imaging revealed the existence of dense granules within the bacterial cells, while EDS identified large amounts of sulfur within them. EDS mapping localized the sulfur to these granules. Subsequent 16S rRNA gene sequencing showed that the bacteria detected in our samples belonged to the genus Chromatium, which are sulfur oxidizers. Thus, EDS mapping made it possible to identify sulfur oxidizers in environmental samples based on localized sulfur within their cells, within a short time (within 24 h of sampling). This technique has wide ranging applications for detection of sulfur bacteria in environmental water samples.

Optimal spatial sampling techniques for ground truth data in microwave remote sensing of soil moisture

NASA Technical Reports Server (NTRS)

Rao, R. G. S.; Ulaby, F. T.

1977-01-01

The paper examines optimal sampling techniques for obtaining accurate spatial averages of soil moisture, at various depths and for cell sizes in the range 2.5-40 acres, with a minimum number of samples. Both simple random sampling and stratified sampling procedures are used to reach a set of recommended sample sizes for each depth and for each cell size. Major conclusions from statistical sampling test results are that (1) the number of samples required decreases with increasing depth; (2) when the total number of samples cannot be prespecified or the moisture in only one single layer is of interest, then a simple random sample procedure should be used which is based on the observed mean and SD for data from a single field; (3) when the total number of samples can be prespecified and the objective is to measure the soil moisture profile with depth, then stratified random sampling based on optimal allocation should be used; and (4) decreasing the sensor resolution cell size leads to fairly large decreases in samples sizes with stratified sampling procedures, whereas only a moderate decrease is obtained in simple random sampling procedures.

Menstrual blood closely resembles the uterine immune micro-environment and is clearly distinct from peripheral blood.

PubMed

van der Molen, R G; Schutten, J H F; van Cranenbroek, B; ter Meer, M; Donckers, J; Scholten, R R; van der Heijden, O W H; Spaanderman, M E A; Joosten, I

2014-02-01

Is menstrual blood a suitable source of endometrial derived lymphocytes? Mononuclear cells isolated from menstrual samples (menstrual blood mononuclear cells (MMC)) are clearly distinct from peripheral blood mononuclear cells (PBMC) and show a strong resemblance with biopsy-derived endometrial mononuclear cells. A critical event in the onset of pregnancy is the implantation of the embryo in the uterine wall. The immune cell composition in the endometrium at the time of implantation is considered pivotal for success. Despite advancing knowledge on the composition of the immune cell population in the uterus, the role of endometrial immune cells in reproductive disorders is still not fully resolved, mainly due to the fact that this type of research requires invasive techniques. Here, we collected menstrual fluid and validated this unique non-invasive technique to obtain and study the endometrium-derived immune cells which would be present around the time of implantation. Five healthy non-pregnant females with regular menstruation cycles and not using oral contraceptives collected their menstrual blood using a menstrual cup in five consecutive cycles. Sampling took place over the first 3 days of menses, with 12 h intervals. Peripheral blood samples, taken before and after each menstruation, were obtained for comparative analysis. MMC and PBMC samples were characterized for the different lymphocyte subsets by flow cytometry, with emphasis on NK cells and T cells. Next, the functional capacity of the MMC-derived NK cells was determined by measuring intracellular production of IFN-γ, granzyme B and perforin after culture in the presence of IL-2 and IL-15. In support of their endometrial origin, MMC samples contained the typical composition of mononuclear cells expected of endometrial tissue, were phenotypically similar to the reported phenotype for biopsy-derived endometrial cells, and were distinct from PBMC. Increased percentages of NK cells and decreased percentages of T cells were found in MMC when compared with PBMC from the same female. The MMC-derived NK cells were pre-dominantly CD56(bright)/CD16(-), in contrast to the primarily CD56(dim)/CD16(+) peripheral blood NK cells. MMC-derived NK cells expressed CD103, indicating their mucosal origin. In addition, the pattern of natural cytotoxicity receptor (NCR) expression in MMC-derived NK cells was comparable with that in endometrial biopsy-derived NK cells. Compared with PBMC, the NKp30 expression was decreased, while the percentage of NKp44 positive cells was increased in MMC samples. CXCR3 and CXCR4 were hardly expressed by MMC-derived NK cells, indicating that these cells are not of PBMC origin. NK cells from MMC samples were functional as shown by their capacity to produce IFN-γ, granzyme B and perforin, upon stimulation with IL-2 and IL-15. MMC-derived T cells revealed an increased expression of CD103, CD69 and CXCR4 compared with PBMC-derived T cells. Importantly, MMC collection using a menstrual cup proved highly reliable and reproducible between women and between cycles. Based on the parameters we studied, MMC appear similar to biopsy-derived endometrial mononuclear cells. However, sampling is not done at the exact same time in the menstrual cycle, and thus we cannot exclude some, as yet undetected, differences. Also, it should be considered that for some women, the use of the menstrual cup may be unpleasant. Menstrual blood may be a source of endometrial cells and may create new opportunities to study uterine immunological cells in fertility issues. No external funding was obtained for the present study. None of the authors have any conflict of interest to declare. NA.

Evaluation of the performance of Human Papillomavirus testing in paired urine and clinician-collected cervical samples among women aged over 30 years in Bhutan.

PubMed

Tshomo, Ugyen; Franceschi, Silvia; Tshokey, Tshokey; Tobgay, Tashi; Baussano, Iacopo; Tenet, Vanessa; Snijders, Peter J F; Gheit, Tarik; Tommasino, Massimo; Vorsters, Alex; Clifford, Gary M

2017-04-08

Urine sampling may offer a less invasive solution than cervical sampling to test for human papillomavirus (HPV) for HPV vaccine impact monitoring. Paired samples of urine and exfoliated cervical cells were obtained for 89 women with history of high-risk (HR) HPV-positive normal cytology in Bhutan. Urine sampling protocol included self-collection of first-void urine immediately into a conservation medium and procedures to optimize DNA yield. Colposcopical abnormalities were biopsied. Two HPV assays were used: a multiplex type-specific PCR (E7-MPG) and a less analytically sensitive GP5+/6+ PCR followed by reverse line blot. HPV positivity for 21 types common to both assays was similar in urine and cells by E7-MPG (62.9% and 57.3%, respectively, p = 0.32) but lower in urine by GP5+/6+ (30.3% and 40.4%, p = 0.05). HPV6/11/16/18 positivity did not significantly differ between urine and cells by either assay. Sensitivity of urine (using cells as gold standard) to detect 21 HPV types was 80% and 58% for E7-MPG and GP5+/6+, respectively, with specificity 61% and 89%. HPV type distribution in urine and cells was similar, regardless of assay. The 5 detected CIN3+ were HR-HPV positive in cells by both assays, compared to 4 and 3 by E7-MPG and GP5+/6+, respectively, in urine samples. For the monitoring of vaccine impact, we demonstrate validity of a urine sampling protocol to obtain HPV prevalence data that are broadly comparable to that from cervical cells. However, detection of HPV in urine varies according to assay sensitivity, presumably because low level infections are frequent.

A multitask clustering approach for single-cell RNA-seq analysis in Recessive Dystrophic Epidermolysis Bullosa

PubMed Central

Petegrosso, Raphael; Tolar, Jakub

2018-01-01

Single-cell RNA sequencing (scRNA-seq) has been widely applied to discover new cell types by detecting sub-populations in a heterogeneous group of cells. Since scRNA-seq experiments have lower read coverage/tag counts and introduce more technical biases compared to bulk RNA-seq experiments, the limited number of sampled cells combined with the experimental biases and other dataset specific variations presents a challenge to cross-dataset analysis and discovery of relevant biological variations across multiple cell populations. In this paper, we introduce a method of variance-driven multitask clustering of single-cell RNA-seq data (scVDMC) that utilizes multiple single-cell populations from biological replicates or different samples. scVDMC clusters single cells in multiple scRNA-seq experiments of similar cell types and markers but varying expression patterns such that the scRNA-seq data are better integrated than typical pooled analyses which only increase the sample size. By controlling the variance among the cell clusters within each dataset and across all the datasets, scVDMC detects cell sub-populations in each individual experiment with shared cell-type markers but varying cluster centers among all the experiments. Applied to two real scRNA-seq datasets with several replicates and one large-scale droplet-based dataset on three patient samples, scVDMC more accurately detected cell populations and known cell markers than pooled clustering and other recently proposed scRNA-seq clustering methods. In the case study applied to in-house Recessive Dystrophic Epidermolysis Bullosa (RDEB) scRNA-seq data, scVDMC revealed several new cell types and unknown markers validated by flow cytometry. MATLAB/Octave code available at https://github.com/kuanglab/scVDMC. PMID:29630593

Method for measuring recovery of catalytic elements from fuel cells

DOEpatents

Shore, Lawrence [Edison, NJ; Matlin, Ramail [Berkeley, NJ

2011-03-08

A method is provided for measuring the concentration of a catalytic clement in a fuel cell powder. The method includes depositing on a porous substrate at least one layer of a powder mixture comprising the fuel cell powder and an internal standard material, ablating a sample of the powder mixture using a laser, and vaporizing the sample using an inductively coupled plasma. A normalized concentration of catalytic element in the sample is determined by quantifying the intensity of a first signal correlated to the amount of catalytic element in the sample, quantifying the intensity of a second signal correlated to the amount of internal standard material in the sample, and using a ratio of the first signal intensity to the second signal intensity to cancel out the effects of sample size.

Comparison of Environmental Scanning Electron Microscopy in Low Vacuum or wet mode for the investigation of cell biomaterial interactions.

PubMed

Mattarozzi, Monica; Manfredi, Edoardo; Lorenzi, Andrea; Smerieri, Arianna; Di Blasio, Alberto; Macaluso, Guido; Lumetti, Simone; Galli, Carlo

2016-05-06

The aim of the present study was to investigate the efficacy of environmental scanning electron microscopy (ESEM), in low vacuum mode (LV-ESEM) and in wet mode (wet-ESEM) in the assessment of cell-material interactions. Mouse calvaria MC3T3 cells (ATCC) were seeded on commercially pure machined titanium discs of 10 mm diameter in Dulbecco modified MEM, 10% Fetal Bovine Serum, 1% Penicillin and Streptomycin and 1% Glutamine. Samples were then processed for microscope observation by rinse in Phosphate Buffer saline and fixation in 4.5% Glutaraldehyde. Samples were then rinsed in Sodium Cacodylate buffer and observed or dehydrated in alcohol prior to LV-ESEM observation. Fresh samples in 0.9% NaCl solution were observed in wet- ESEM. No significant loss of detail was observed when dehydrated or non dehydrated samples were analysed at LV-ESEM.The observation of fresh samples in wet-ESEM however proved difficult for the need to eliminate water which forms a layer covering the sample, thus hiding cell surface details. When reducing the vapor pressure in the chamber, the layer evaporated and NaCl immediately started to precipitate and cells collapsed, thus no further investigation was possible. The use of low vacuum-ESEM after cell fixation, but without dehydration or gold sputter coating proved a viable alternative to traditional high vacuum SEM observation.

The effect of sunshine testing on terrestrial solar cell system components

NASA Technical Reports Server (NTRS)

Forestieri, A. F.; Anagnostou, E.

1975-01-01

Samples of FEP encapsulated silicon solar cells and various potential encapsulation or cover materials were subjected to accelerated and real time testing. By measuring changes in solar cell output or optical transmission as a function of exposure the durability of the samples was evaluated. Results are presented.

Use Of The Diamond Cell In An Industrial Laboratory

NASA Astrophysics Data System (ADS)

Barbour, Rachael L.; Stephens, J. D.; Cameron, David G.

1989-12-01

The traditional method for recording the IR spectra of solids has been KBr pellet transmission spectroscopy. This technique has several disadvantages: sample preparation time, matrix contamination, spectral distortion, ion exchange, a limited spectral range, scattering, loss of sample integrity during grinding, etc. In recent years, diffuse reflectance, ATR, photoacoustic reflectance, and external reflectance have been used increasingly, facilitated by the high SNR of FT instruments. In many cases, the diamond cell is an attractive alternative to all of these. The spectral range is -100 -1 to the UV, excluding the 2200-2000 cm -1 region. Spectral distortion, usually a great problem with inorganics, is greatly reduced as a result of sample homogeneity (from a spectral point of view) and refractive index matching. There is no matrix contamination: scattering, background slope, and all absorption bands are from the sample. There is no ion exchange. The sample size requirements are minimal. Finally, sample preparation requires the somewhat lost. but powerful, art of microscopic examination. In some instances, there may be sample orientation or pressure induced phase changes associated with the use of the diamond cell. A common misconception is that an IR microscope is needed to use the diamond cell. In fact, ~5 minutes will suffice without a beam condenser; 1 minute is all that is needed with one. In part, this is because one usually has excellent control of the optical thickness; with experience, the cell can easily be assembled to give bands in the 0.7-1.5 absorbance range, and making the sample thinner merely involves pressing the diamonds together. Given the above, the microscope should only be used for inhomogeneous samples as one loses all information below 700 cm-1, the region of greatest value when studying inorganics. We also note that the cell can readily be moved from a mid-IR to a far-IR bench. We have moved to the point where this is the dominant sampling technique, with ATR being the next most important. Diffuse reflectance and KBr pellets are seldom used. The cell has been used on inorganics (mid and far IR) including extremely small pure mineral samples selected by hand. It is also used for polymers, polymer inclusions, filter deposits, pure (and not so puce) organics, and general "what is this stuff" samples. Examples of a wide variety of analyses will be given.

Isolation of Circulating Plasma Cells in Multiple Myeloma Using CD138 Antibody-Based Capture in a Microfluidic Device

NASA Astrophysics Data System (ADS)

Qasaimeh, Mohammad A.; Wu, Yichao C.; Bose, Suman; Menachery, Anoop; Talluri, Srikanth; Gonzalez, Gabriel; Fulciniti, Mariateresa; Karp, Jeffrey M.; Prabhala, Rao H.; Karnik, Rohit

2017-04-01

The necessity for bone marrow aspiration and the lack of highly sensitive assays to detect residual disease present challenges for effective management of multiple myeloma (MM), a plasma cell cancer. We show that a microfluidic cell capture based on CD138 antigen, which is highly expressed on plasma cells, permits quantitation of rare circulating plasma cells (CPCs) in blood and subsequent fluorescence-based assays. The microfluidic device is based on a herringbone channel design, and exhibits an estimated cell capture efficiency of ~40-70%, permitting detection of <10 CPCs/mL using 1-mL sample volumes, which is difficult using existing techniques. In bone marrow samples, the microfluidic-based plasma cell counts exhibited excellent correlation with flow cytometry analysis. In peripheral blood samples, the device detected a baseline of 2-5 CD138+ cells/mL in healthy donor blood, with significantly higher numbers in blood samples of MM patients in remission (20-24 CD138+ cells/mL), and yet higher numbers in MM patients exhibiting disease (45-184 CD138+ cells/mL). Analysis of CPCs isolated using the device was consistent with serum immunoglobulin assays that are commonly used in MM diagnostics. These results indicate the potential of CD138-based microfluidic CPC capture as a useful ‘liquid biopsy’ that may complement or partially replace bone marrow aspiration.

Mammary stem cell and macrophage markers are enriched in normal tissue adjacent to inflammatory breast cancer.

PubMed

Reddy, Jay P; Atkinson, Rachel L; Larson, Richard; Burks, Jared K; Smith, Daniel; Debeb, Bisrat G; Ruffell, Brian; Creighton, Chad J; Bambhroliya, Arvind; Reuben, James M; Van Laere, Steven J; Krishnamurthy, Savitri; Symmans, William F; Brewster, Abenaa M; Woodward, Wendy A

2018-06-01

We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. Normal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44 + CD49f + CD133/2 + mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. 8 of 8 IBC samples expressed isolated CD44 + CD49f + CD133/2 + stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44 + CD49f + CD133/2 + cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001). Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

Broadband thermal optical limiter for the protection of eyes and sensors

NASA Astrophysics Data System (ADS)

Justus, Brian L.; Huston, Alan L.; Campillo, Anthony J.

1994-05-01

A broadband thermal optical limiter for protecting a light sensitive object from intense laser beams at all near ultraviolet, visible and near infrared wavelengths is disclosed. The broadband thermal optical limiter comprises: a sample cell containing a solution of broadband absorber material dissolved in a thermal solvent; and a first optical device for converging an incident laser beam into the sample cell. The sample cell is responsive to a converged incident laser beam below a predetermined intensity level for passing therethrough the converged incident laser beam below the predetermined intensity level. The sample cell is also responsive to a converged incident laser beam at or above a predetermined intensity level for thermally defocusing substantially all of the converged incident laser beam in different directions and passing therethrough only a remaining small portion of the converged incident laser beam at or above the predetermined intensity level. The broadband thermal optical limiter further includes a second optical device for focusing substantially all of the laser beam passing through the sample cell into the light sensitive object to be protected.

Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates.

PubMed Central

Forus, A; Høifødt, H K; Overli, G E; Myklebost, O; Fodstad, O

1999-01-01

AIM: The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. METHODS: In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. RESULTS: In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10(7). Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10(7) mononuclear cells, respectively. The patient with 540 tumour cells/10(7) mononuclear cells showed high level amplification of erbB2 and suffered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10(7) mononuclear cells had favourable disease progression. CONCLUSION: These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information. PMID:10474684

Multivariate classification of infrared spectra of cell and tissue samples

DOEpatents

Haaland, David M.; Jones, Howland D. T.; Thomas, Edward V.

1997-01-01

Multivariate classification techniques are applied to spectra from cell and tissue samples irradiated with infrared radiation to determine if the samples are normal or abnormal (cancerous). Mid and near infrared radiation can be used for in vivo and in vitro classifications using at least different wavelengths.

Immunohistochemical and morphometric analysis of intestinal full-thickness biopsy samples from cats with lymphoplasmacytic inflammatory bowel disease.

PubMed

Marsilio, S; Kleinschmidt, S; Nolte, I; Hewicker-Trautwein, M

2014-05-01

The distribution and numbers of CD3(+) T lymphocytes, immunoglobulin(+) plasma cells and calprotectin (L1)(+) macrophages was analyzed in full-thickness, formalin-fixed biopsy samples from the small intestine (duodenum, jejunum and ileum) and from the colon from nine cats with clinical signs of inflammatory bowel disease (IBD). All animals had lymphoplasmacytic enteritis or lymphoplasmacytic enterocolitis. Equivalent samples from the same intestinal regions from 12 healthy pet cats served as controls. Labelled cells in the lamina propria were counted by computer-aided morphometry. The different cell types were similarly distributed in both groups, but there were differences in their numbers. There were more CD3(+) T cells in the duodenum and jejunum of cats with IBD; however, the difference was only significant for the duodenum. There were significantly more IgA(+) cells in the duodenal crypt region. There were significantly more IgG(+) cells in the lower jejunal crypt region. Plasma cells expressing IgM were decreased in cats with IBD, but the difference was not significant. L1(+) macrophages were significantly decreased in the lower crypt area of the colon in cats with IBD and there was a trend to decreased L1(+) cells in the upper crypt area of the duodenum and jejunum. Comparison of the results of this study with previous findings on endoscopically-obtained duodenal biopsy samples from cats with IBD revealed some differences. These discrepancies might relate to differences between control cat populations, types of biopsy samples, methodological factors such as different counting techniques and the activity of the disease at the time of sampling. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enhanced detection of infectious hematopoietic necrosis virus by pretreatment of cell monolayers with polyethylene glycol

USGS Publications Warehouse

Batts, W.N.; Winton, J.R.

1989-01-01

To improve quantification of very low levels of infectious hematopoietic necrosis virus (IHNV) in samples of tissue, ovarian fluid, or natural water supplies, we tested the ability of polyethylene glycol (PEG) to enhance the sensitivity and speed of the plaque assay system. We compared 4, 7, and 10% solutions of PEG of molecular weight 6,000, 8,000, or 20,000 applied at selected volumes and for various durations. When cell monolayers of epithelioma papulosum cyprini (EPC), fathead minnow (FHM), chinook salmon embryo (CHSE-214), and bluegill fry (BF2) were pretreated with 7% PEG-20,000, they produced 4-17-fold increases in plaque assay titers of IHNV. The plaque assay titers of viral hemorrhagic septicemia virus, chum salmon reovirus, and chinook salmon paramyxovirus were also enhanced by exposure of CHSE-214 cells to PEG, but the titers of infectious pancreatic necrosis virus and Oncorhynchus masou virus were not substantially changed. Plaques formed by IHNV on PEG-treated EPC cells incubated at 15°C had a larger mean diameter at 6 d than those on control cells at 8 d; this suggests the assay could be shortened by use of PEG. Pretreatment of EPC cell monolayers with PEG enabled detection of IHNV in some samples that appeared negative with untreated cells. For example, when ovarian fluid samples from chinook salmon Oncorhynchus tshawytscha were inoculated onto untreated monolayers of EPC cells, IHNV was detected in only 11 of 51 samples; 17 of the samples were positive when PEG-treated EPC cells were used.PDF

Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results

PubMed Central

Bordag, Natalie; Janakiraman, Vijay; Nachtigall, Jonny; González Maldonado, Sandra; Bethan, Bianca; Laine, Jean-Philippe; Fux, Elie

2016-01-01

The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench) followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli) as well as mammalian cells chinese hamster ovary (CHO) and mouse myeloma cells (NS0).The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data. PMID:27438065

The magnetic introduction of magnetite nanoparticles into live cells for radiosensibility enhancement

NASA Astrophysics Data System (ADS)

Yurenya, Anton Y.; Polikarpov, Mikhail A.; Chukalova, Aynur A.; Moskaleva, Elizaveta Y.; Taldenkov, Alexander N.; Panchenko, Vladislav Y.

2017-04-01

Earlier we proposed a new radiotherapy enhancement method that entails the administration of 57Fe iron-oxide nanoparticles into the cells [5]. Within this work we were prompt to investigate the capability of iron oxide nanoparticles with monolayer coating to penetrate into live cells. Magnetite particle samples were synthesized and stabilized with HCl or citric acid. The cells were incubated in the presence of nanoparticles for 1 h, washed and dried. To distinguish inside-cell particles from outside ones a set of experiments with low temperature incubation was carried out. Several cell samples were prepared in the presence of an external magnetic field in order to study the possibility of the nanoparticle uptake enhancement. To evaluate the amount of particles in each cell sample we used a SQUID-magnetometer. The nanoparticle suspension with HCl stabilization turned to be inadequate for intracellular introduction. Approximately 2·105 particles with citric acid covering conjugated with each cell after incubation at normal conditions. An application of an external magnetic field increased this amount up to 107 particles/cell. Most probably much of these particles penetrated into cells.

Multilaser Herriott Cell for Planetary Tunable Laser Spectrometers

NASA Technical Reports Server (NTRS)

Tarsitano, Christopher G.; Webster, Christopher R.

2007-01-01

Geometric optics and matrix methods are used to mathematically model multilaser Herriott cells for tunable laser absorption spectrometers for planetary missions. The Herriott cells presented accommodate several laser sources that follow independent optical paths but probe a single gas cell. Strategically placed output holes located in the far mirrors of the Herriott cells reduce the size of the spectrometers. A four-channel Herriott cell configuration is presented for the specific application as the sample cell of the tunable laser spectrometer instrument selected for the sample analysis at Mars analytical suite on the 2009 Mars Science Laboratory mission.

Single-cell transcriptome conservation in cryopreserved cells and tissues.

PubMed

Guillaumet-Adkins, Amy; Rodríguez-Esteban, Gustavo; Mereu, Elisabetta; Mendez-Lago, Maria; Jaitin, Diego A; Villanueva, Alberto; Vidal, August; Martinez-Marti, Alex; Felip, Enriqueta; Vivancos, Ana; Keren-Shaul, Hadas; Heath, Simon; Gut, Marta; Amit, Ido; Gut, Ivo; Heyn, Holger

2017-03-01

A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.

A Novel Technique for Performing PID Susceptibility Screening during the Solar Cell Fabrication Process

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Jaewon; Dahal, Som; Dauksher, Bill

2016-11-21

Various characterization techniques have historically been developed in order to screen potential induced degradation (PID)-susceptible cells, but those techniques require final solar cells. We present a new characterization technique for screening PID-susceptible cells during the cell fabrication process. Illuminated Lock-In Thermography (ILIT) was used to image PID shunting of the cell without metallization and clearly showed PID-affected areas. PID-susceptible cells can be screened by ILIT, and the sample structure can advantageously be simplified as long as the sample has the silicon nitride antireflection coating and an aluminum back surface field.

Intra-Hepatic Depletion of Mucosal-Associated Invariant T Cells in Hepatitis C Virus-Induced Liver Inflammation.

PubMed

Bolte, Fabian J; O'Keefe, Ashley C; Webb, Lauren M; Serti, Elisavet; Rivera, Elenita; Liang, T Jake; Ghany, Marc; Rehermann, Barbara

2017-11-01

Chronic hepatitis affects phenotypes of innate and adaptive immune cells. Mucosal-associated invariant T (MAIT) cells are enriched in the liver as compared with the blood, respond to intra-hepatic cytokines, and (via the semi-invariant T-cell receptor) to bacteria translocated from the gut. Little is known about the role of MAIT cells in livers of patients with chronic hepatitis C virus (HCV) infection and their fate after antiviral therapy. We collected blood samples from 42 patients with chronic HCV infection who achieved a sustained virologic response after 12 weeks of treatment with sofosbuvir and velpatasvir. Mononuclear cells were isolated from blood before treatment, at weeks 4 and 12 during treatment, and 24 weeks after the end of treatment. Liver biopsies were collected from 37 of the patients prior to and at week 4 of treatment. Mononuclear cells from 56 blood donors and 10 livers that were not suitable for transplantation were used as controls. Liver samples were assessed histologically for inflammation and fibrosis. Mononuclear cells from liver and blood were studied by flow cytometry and analyzed for responses to cytokine and bacterial stimulation. The frequency of MAIT cells among T cells was significantly lower in blood and liver samples of patients with HCV infection than of controls (median, 1.31% vs 2.32% for blood samples, P = .0048; and median, 4.34% vs 13.40% for liver samples, P = .001). There was an inverse correlation between the frequency of MAIT cells in the liver and histologically determined levels of liver inflammation (r = -.5437, P = .0006) and fibrosis (r = -.5829, P = .0002). MAIT cells from the liver had higher levels of activation and cytotoxicity than MAIT cells from blood (P < .0001). Production of interferon gamma by MAIT cells was dependent on monocyte-derived interleukin 18, and was reduced in patients with HCV infection in response to T-cell receptor-mediated but not cytokine-mediated stimulation, as compared with controls. Anti-viral therapy rapidly decreased liver inflammation and MAIT cell activation and cytotoxicity, and increased the MAIT cell frequency among intra-hepatic but not blood T cells. The MAIT cell response to T-cell receptor-mediated stimulation did not change during the 12 weeks of antiviral therapy. In analyses of paired blood and liver samples from patients with chronic HCV infection before, during, and after antiviral therapy with sofosbuvir and velpatasvir, we found that intrahepatic MAIT cells are activated by monocyte-derived cytokines and depleted in HCV-induced liver inflammation. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Single cell network profiling assay in bladder cancer.

PubMed

Covey, Todd M; Vira, Manish A; Westfall, Matt; Gulrajani, Michael; Cholankeril, Michelle; Okhunov, Zhamshid; Levey, Helen R; Marimpietri, Carol; Hawtin, Rachael; Fields, Scott Z; Cesano, Alessandra

2013-04-01

The aim of this study was to assess the feasibility of applying the single cell network profiling (SCNP) assay to the examination of signaling networks in epithelial cancer cells, using bladder washings from 29 bladder cancer (BC) and 15 nonbladder cancer (NC) subjects. This report describes the methods we developed to detect rare epithelial cells (within the cells we collected from bladder washings), distinguish cancer cells from normal epithelial cells, and reproducibly quantify signaling within these low frequency cancer cells. Specifically, antibodies against CD45, cytokeratin, EpCAM, and cleaved-PARP (cPARP) were used to differentiate nonapoptotic epithelial cells from leukocytes, while measurements of DNA content to determine aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p-AKT and p-ERK at baseline and in response to pathway modulation; 66% (N = 19) of BC samples and 27% (N = 4) of NC samples met the "evaluable" criteria, i.e., at least 400,000 total cells available upon sample receipt with >2% of cells showing an epithelial phenotype. The majority of epithelial cells detected in BC samples were nonapoptotic and all signaling data were generated from identified cPARP negative cells. In four of 19 BC samples but in none of the NC specimens, SCNP assay identified epithelial cancer cells with a quantifiable increase in epidermal growth factor-induced p-AKT and p-ERK levels. Furthermore, preincubation with the PI3K inhibitor GDC-0941 reduced or completely inhibited basal and epidermal growth factor-induced p-AKT but, as expected, had no effect on p-ERK levels. This study demonstrates the feasibility of applying SCNP assay using multiparametric flow cytometry to the functional characterization of rare, bladder cancer cells collected from bladder washing. Following assay standardization, this method could potentially serve as a tool for disease characterization and drug development in bladder cancer and other solid tumors. Copyright © 2013 International Society for Advancement of Cytometry.

Evaluating hepatocellular carcinoma cell lines for tumour samples using within-sample relative expression orderings of genes.

PubMed

Ao, Lu; Guo, You; Song, Xuekun; Guan, Qingzhou; Zheng, Weicheng; Zhang, Jiahui; Huang, Haiyan; Zou, Yi; Guo, Zheng; Wang, Xianlong

2017-11-01

Concerns are raised about the representativeness of cell lines for tumours due to the culture environment and misidentification. Liver is a major metastatic destination of many cancers, which might further confuse the origin of hepatocellular carcinoma cell lines. Therefore, it is of crucial importance to understand how well they can represent hepatocellular carcinoma. The HCC-specific gene pairs with highly stable relative expression orderings in more than 99% of hepatocellular carcinoma but with reversed relative expression orderings in at least 99% of one of the six types of cancer, colorectal carcinoma, breast carcinoma, non-small-cell lung cancer, gastric carcinoma, pancreatic carcinoma and ovarian carcinoma, were identified. With the simple majority rule, the HCC-specific relative expression orderings from comparisons with colorectal carcinoma and breast carcinoma could exactly discriminate primary hepatocellular carcinoma samples from both primary colorectal carcinoma and breast carcinoma samples. Especially, they correctly classified more than 90% of liver metastatic samples from colorectal carcinoma and breast carcinoma to their original tumours. Finally, using these HCC-specific relative expression orderings from comparisons with six cancer types, we identified eight of 24 hepatocellular carcinoma cell lines in the Cancer Cell Line Encyclopedia (Huh-7, Huh-1, HepG2, Hep3B, JHH-5, JHH-7, C3A and Alexander cells) that are highly representative of hepatocellular carcinoma. Evaluated with a REOs-based prognostic signature for hepatocellular carcinoma, all these eight cell lines showed the same metastatic properties of the high-risk metastatic hepatocellular carcinoma tissues. Caution should be taken for using hepatocellular carcinoma cell lines. Our results should be helpful to select proper hepatocellular carcinoma cell lines for biological experiments. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Heterogeneous, restricted patterns of Epstein-Barr virus (EBV) latent gene expression in patients with chronic active EBV infection.

PubMed

Yoshioka, M; Ishiguro, N; Ishiko, H; Ma, X; Kikuta, H; Kobayashi, K

2001-10-01

Epstein-Barr virus (EBV) has been shown to infect T lymphocytes and to be associated with a chronic active infection (CAEBV), which has been recognized as a mainly non-neoplastic T-cell lymphoproliferative disorder (T-cell LPD). The systemic distribution of EBV genomes was studied, by real-time PCR, in multiple tissues from six patients with CAEBV, including three patients with T-cell LPD, one patient with B-cell LPD and two patients with undetermined cell-type LPD. There were extremely high loads of EBV genomes in all tissues from the patients. This reflects an abundance of circulating and infiltrating EBV-infected cells and a wide variety of clinical symptoms in the affected tissues. We chose one sample from each patient that was shown by real-time PCR to contain a high load of EBV genomes and examined the expression of EBV latent genes by RT-PCR. EBER1 and EBNA1 transcripts were detected in all samples. Only one sample also expressed EBNA2, LMP1 and LMP2A transcripts in addition to EBER1 and EBNA1 transcripts. Two of the remaining five samples expressed LMP1 and LMP2A transcripts. One sample expressed LMP2A but not LMP1 and EBNA2 transcripts. Another sample expressed EBNA2 but not LMP1 and LMP2A transcripts. The other sample did not express transcripts of any of the other EBNAs or LMPs. None of the samples expressed the viral immediate-early gene BZLF1. These results showed that EBV latent gene expression in CAEBV is heterogeneous and that restricted forms of EBV latency might play a pathogenic role in the development of CAEBV.

A Novel 96well-formatted Micro-gap Plate Enabling Drug Response Profiling on Primary Tumour Samples

NASA Astrophysics Data System (ADS)

Ma, Wei-Yuan; Hsiung, Lo-Chang; Wang, Chen-Ho; Chiang, Chi-Ling; Lin, Ching-Hung; Huang, Chiun-Sheng; Wo, Andrew M.

2015-04-01

Drug-based treatments are the most widely used interventions for cancer management. Personalized drug response profiling remains inherently challenging with low cell count harvested from tumour sample. We present a 96well-formatted microfluidic plate with built-in micro-gap that preserves up to 99.2% of cells during multiple assay/wash operation and only 9,000 cells needed for a single reagent test (i.e. 1,000 cells per test spot x 3 selected concentration x triplication), enabling drug screening and compatibility with conventional automated workstations. Results with MCF7 and MDA-MB-231 cell lines showed that no statistical significance was found in dose-response between the device and conventional 96-well plate control. Primary tumour samples from breast cancer patients tested in the device also showed good IC50 prediction. With drug screening of primary cancer cells must consider a wide range of scenarios, e.g. suspended/attached cell types and rare/abundant cell availability, the device enables high throughput screening even for suspended cells with low cell count since the signature microfluidic cell-trapping feature ensures cell preservation in a multiple solution exchange protocol.

A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry

NASA Astrophysics Data System (ADS)

Patibandla, Phani K.; Estrada, Rosendo; Kannan, Manasaa; Sethu, Palaniappan

2014-03-01

Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (˜45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain ‘analysis-ready’ samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ‘holding time’ to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the conventional procedure (45 min) and our microfluidic approach (12 min).

Overexpression of syndecan-1, MUC-1, and putative stem cell markers in breast cancer leptomeningeal metastasis: a cerebrospinal fluid flow cytometry study.

PubMed

Cordone, Iole; Masi, Serena; Summa, Valentina; Carosi, Mariantonia; Vidiri, Antonello; Fabi, Alessandra; Pasquale, Alessia; Conti, Laura; Rosito, Immacolata; Carapella, Carmine Maria; Villani, Veronica; Pace, Andrea

2017-04-11

Cancer is a mosaic of tumor cell subpopulations, where only a minority is responsible for disease recurrence and cancer invasiveness. We focused on one of the most aggressive circulating tumor cells (CTCs) which, from the primitive tumor, spreads to the central nervous system (CNS), evaluating the expression of prognostic and putative cancer stem cell markers in breast cancer (BC) leptomeningeal metastasis (LM). Flow cytometry immunophenotypic analysis of cerebrospinal fluid (CSF) samples (4.5 ml) was performed in 13 consecutive cases of BCLM. Syndecan-1 (CD138), MUC-1 (CD227) CD45, CD34, and the putative cancer stem cell markers CD15, CD24, CD44, and CD133 surface expression were evaluated on CSF floating tumor cells. The tumor-associated leukocyte population was also characterized. Despite a low absolute cell number (8 cell/μl, range 1-86), the flow cytometry characterization was successfully conducted in all the samples. Syndecan-1 and MUC-1 overexpression was documented on BC cells in all the samples analyzed; CD44, CD24, CD15, and CD133 in 77%, 75%, 70%, and 45% of cases, respectively. A strong syndecan-1 and MUC-1 expression was also documented by immunohistochemistry on primary breast cancer tissues, performed in four patients. The CSF tumor population was flanked by T lymphocytes, with a different immunophenotype between the CSF and peripheral blood samples (P ≤ 0.02). Flow cytometry can be successfully employed for solid tumor LM characterization even in CSF samples with low cell count. This in vivo study documents that CSF floating BC cells overexpress prognostic and putative cancer stem cell biomarkers related to tumor invasiveness, potentially representing a molecular target for circulating tumor cell detection and LM treatment monitoring, as well as a primary target for innovative treatment strategies. The T lymphocyte infiltration, documented in all CSF samples, suggests a possible involvement of the CNS lymphatic system in both lymphoid and cancer cell migration into and out of the meninges, supporting the extension of a new form of cellular immunotherapy to LM. Due to the small number of cases, validation on large cohorts of patients are warranted to confirm these findings and to evaluate the impact and value of these results for diagnosis and management of LM.

Correlation between surface carbon concentration and adhesive strength at the Si cell/EVA interface in a PV module

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhere, N.G.; Wollam, M.E.; Gadre, K.S.

1997-12-31

Silicon solar cell/EVA composite is being studied with an objective to further improve the manufacturing technology of PV modules. Sample extraction and adhesion strength measurement process has been modified. Silicon and EVA samples were extracted from solar cells of new and field-deployed modules. Optical microscopy, SEM, and AES of samples from new modules revealed EVA islands covering most of the silicon cell surface indicating a cohesive failure. A good correlation was observed between the adhesive strength and surface concentration of carbon. A low carbon concentration which indicated less EVA clinging to cell surface always resulted in low adhesive strengths. Themore » correlation provides a simple technique for inferring properties of EVA.« less

NASA welding assessment program

NASA Technical Reports Server (NTRS)

Patterson, R. E.

1985-01-01

A program was conducted to demonstrate the cycle life capability of welded solar cell modules relative to a soldered solar cell module in a simulated low earth orbit thermal environment. A total of five 18-cell welded (parallel gap resistance welding) modules, three 18-cell soldered modules, and eighteen single cell samples were fabricated using 2 x 4 cm silicon solar cells from ASEC, fused silica cover glass from OCLI, silver plated Invar interconnectors, DC 93-500 adhesive, and Kapton-Kevlar-Kapton flexible substrate material. Zero degree pull strength ranged from 2.4 to 5.7 lbs for front welded contacts (40 samples), and 3.5 to 6.2 lbs for back welded contacts (40 samples). Solar cell cross sections show solid state welding on both front and rear contacts. The 18-cell welded modules have a specific power of 124 W/kg and an area power density of 142 W/sq m (both at 28 C). Three welded and one soldered module were thermal cycle tested in a thermal vacuum chamber simulating a low earth orbit thermal environment.

FISH-in-CHIPS: A Microfluidic Platform for Molecular Typing of Cancer Cells.

PubMed

Perez-Toralla, Karla; Mottet, Guillaume; Tulukcuoglu-Guneri, Ezgi; Champ, Jérôme; Bidard, François-Clément; Pierga, Jean-Yves; Klijanienko, Jerzy; Draskovic, Irena; Malaquin, Laurent; Viovy, Jean-Louis; Descroix, Stéphanie

2017-01-01

Microfluidics offer powerful tools for the control, manipulation, and analysis of cells, in particular for the assessment of cell malignancy or the study of cell subpopulations. However, implementing complex biological protocols on chip remains a challenge. Sample preparation is often performed off chip using multiple manually performed steps, and protocols usually include different dehydration and drying steps that are not always compatible with a microfluidic format.Here, we report the implementation of a Fluorescence in situ Hybridization (FISH) protocol for the molecular typing of cancer cells in a simple and low-cost device. The geometry of the chip allows integrating the sample preparation steps to efficiently assess the genomic content of individual cells using a minute amount of sample. The FISH protocol can be fully automated, thus enabling its use in routine clinical practice.

System and method for detecting cells or components thereof

DOEpatents

Porter, Marc D [Ames, IA; Lipert, Robert J [Ames, IA; Doyle, Robert T [Ames, IA; Grubisha, Desiree S [Corona, CA; Rahman, Salma [Ames, IA

2009-01-06

A system and method for detecting a detectably labeled cell or component thereof in a sample comprising one or more cells or components thereof, at least one cell or component thereof of which is detectably labeled with at least two detectable labels. In one embodiment, the method comprises: (i) introducing the sample into one or more flow cells of a flow cytometer, (ii) irradiating the sample with one or more light sources that are absorbed by the at least two detectable labels, the absorption of which is to be detected, and (iii) detecting simultaneously the absorption of light by the at least two detectable labels on the detectably labeled cell or component thereof with an array of photomultiplier tubes, which are operably linked to two or more filters that selectively transmit detectable emissions from the at least two detectable labels.

Self-contained high-pressure cell, apparatus, and procedure for the preparation of encapsulated proteins dissolved in low viscosity fluids for nuclear magnetic resonance spectroscopy

NASA Astrophysics Data System (ADS)

Peterson, Ronald W.; Wand, A. Joshua

2005-09-01

The design of a sample cell for high-performance nuclear magnetic resonance (NMR) at elevated pressure is described. The cell has been optimized for the study of encapsulated proteins dissolved in low viscosity fluids but is suitable for more general nuclear magnetic resonance (NMR) spectroscopy of biomolecules at elevated pressure. The NMR cell is comprised of an alumina-toughened zirconia tube mounted on a self-sealing nonmagnetic metallic valve. The cell has several advantages, including relatively low cost, excellent NMR performance, high-pressure tolerance, chemical inertness, and a relatively large active volume. Also described is a low volume sample preparation device that allows for the preparation of samples under high hydrostatic pressure and their subsequent transfer to the NMR cell.

Relationships between host and symbiont cell cycles in sea anemones and their symbiotic dinoflagellates.

PubMed

Dimond, James L; Pineda, Rea R; Ramos-Ascherl, Zullaylee; Bingham, Brian L

2013-10-01

The processes by which cnidarians and their algal endosymbionts achieve balanced growth and biomass could include coordination of host and symbiont cell cycles. We evaluated this theory with natural populations of sea anemones hosting symbiotic dinoflagellates, focusing on the temperate sea anemone Anthopleura elegantissima symbiotic with Symbiodinium muscatinei in Washington State, USA, and the tropical anemone Stichodactyla helianthus associating with unknown Symbiodinium spp. in Belize. By extruding symbiont-containing gastrodermal cells from the relatively large tentacles of these species and using nuclear staining and flow cytometry, we selectively analyzed cell cycle distributions of the symbionts and the host gastrodermal cells that house them. We found no indications of diel synchrony in host and symbiont G2/M phases, and we observed evidence of diel periodicity only in Symbiodinium spp. associated with S. helianthus but not in the anemone itself. Seasonally, S. muscatinei showed considerable G2/M phase variability among samples collected quarterly over an annual period, while the G2/M phase of its host varied much less. Within samples taken at different times of the year, correlations between host and symbiont G2/M phases ranged from very weakly to very strongly positive, with significant correlations in only half of the samples (two of four A. elegantissima samples and one of two S. helianthus samples). Overall, the G2/M phase relationships across species and sampling periods were positive. Thus, while we found no evidence of close cell cycle coupling, our results suggest a loose, positive relationship between cell cycle processes of the symbiotic partners.

Anti-glucagon-like peptide-1 immunoreactivity in samples of blood and ileum obtained from neonatal and adult alpacas.

PubMed

Smith, Courtney C; Cebra, Christopher K; Heidel, Jerry R; Stang, Bernadette V

2013-11-01

To compare numbers of L cells in intestinal samples and blood concentrations of glucagon-like peptide (GLP)-1 between neonatal and mature alpacas. Intestinal samples from carcasses of 4 suckling crias and 4 postweaning alpacas for immunohistochemical analysis and blood samples from 32 suckling crias and 19 healthy adult alpacas for an ELISA. Immunohistochemical staining was conducted in accordance with Oregon State University Veterinary Diagnostic Laboratory standard procedures with a rabbit polyclonal anti-GLP-1 primary antibody. Stained cells with staining results in ileal tissue were counted in 20 fields by 2 investigators, and the mean value was calculated. For quantification of GLP-1 concentrations, blood samples were collected into tubes containing a dipeptidyl peptidase-4 inhibitor. Plasma samples were tested in duplicate with a commercial GLP-1 ELISA validated for use in alpacas. Counts of stained cells (mean ± SD, 50 ± 18 cells) and plasma GLP-1 concentrations (median, 0.086 ng/mL; interquartile range, 0.061 to 0.144 ng/mL) were higher for suckling alpacas than for postsuckling alpacas (stained cells, 26 ± 4 cells; plasma GLP-1 concentration, median, 0.034 ng/mL; interquartile range, 0.015 to 0.048 ng/mL). Older alpacas had lower numbers of L cells in intestinal tissues and lower blood concentrations of GLP-1 than those in neonates. These findings suggested that there may be a decrease in the contribution of GLP-1 to insulin production in adult alpacas, compared with the contribution in neonates.

Investigation of New Semiinsulating Behavior of III-V Compounds.

DTIC Science & Technology

1990-02-23

load (I 10) directions, respectively. Open circles correspond to p-type samples cell . The sample with the length Io of 7 mm, was placed deformed in the...DISCUSSION at a constant rate dl /dt of 0.05 mm/min. The load cell was used to monitor the applied force. All samples used in this A. Free-carrier...the growth of epitaxial quality GaAs bulk crystals (Bryskiewicz et al 1987b). A schematic diagram of the growth cell used in our growth experi- S-nts

Data Validation Package August 2015 Groundwater Sampling at the Grand Junction, Colorado, Disposal Site October 2015

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frazier, William; Baur, Gary

Sampling Period: August 4, 2015. The 1998 Interim Long-Term Surveillance Plan for the Cheney Disposal Site Near Grand Junction, Colorado, requires annual monitoring to assess the performance of the disposal cell. Monitoring wells 0731, 0732, and 0733 were sampled as specified in the plan. Sampling and analyses were conducted in accordance with Sampling and Analysis Plan for the U.S. Department of Energy Office of Legacy Management Sites (LMS/PRO/S04351, continually updated). The water level was measured at each sampled well. The water level in well 0733, located in the disposal cell, is lower than water levels in adjacent wells 0731 andmore » 0732, indicating a hydraulic gradient toward the disposal cell. Results from this sampling event were generally consistent with results from the past as shown in the attached concentration-versus-time graphs. There have been no large changes in contaminant concentration observed over the last several years with the following exception. The uranium concentration in well 0733 has been trending upward since 2003. High uranium concentrations are expected in this well because it is located in the disposal cell. The selenium concentrations observed in wells 0731 and 0732 are elevated when compared to the disposal cell 0733. Wells 0731 and 0732 are completed at the alluvium/Mancos contact; here, elevated selenium concentrations are expected due to contributions from the Mancos shale.« less

Evaluation of the performance of a point-of-care method for total and differential white blood cell count in clozapine users.

PubMed

Bui, H N; Bogers, J P A M; Cohen, D; Njo, T; Herruer, M H

2016-12-01

We evaluated the performance of the HemoCue WBC DIFF, a point-of-care device for total and differential white cell count, primarily to test its suitability for the mandatory white blood cell monitoring in clozapine use. Leukocyte count and 5-part differentiation was performed by the point-of-care device and by routine laboratory method in venous EDTA-blood samples from 20 clozapine users, 20 neutropenic patients, and 20 healthy volunteers. From the volunteers, also a capillary sample was drawn. Intra-assay reproducibility and drop-to-drop variation were tested. The correlation between both methods in venous samples was r > 0.95 for leukocyte, neutrophil, and lymphocyte counts. The correlation between point-of-care (capillary sample) and routine (venous sample) methods for these cells was 0.772; 0.817 and 0.798, respectively. Only for leukocyte and neutrophil counts, the intra-assay reproducibility was sufficient. The point-of-care device can be used to screen for leukocyte and neutrophil counts. Because of the relatively high measurement uncertainty and poor correlation with venous samples, we recommend to repeat the measurement with a venous sample if cell counts are in the lower reference range. In case of clozapine therapy, neutropenia can probably be excluded if high neutrophil counts are found and patients can continue their therapy. © 2016 John Wiley & Sons Ltd.

Mapping cell populations in flow cytometry data for cross‐sample comparison using the Friedman–Rafsky test statistic as a distance measure

PubMed Central

Hsiao, Chiaowen; Liu, Mengya; Stanton, Rick; McGee, Monnie; Qian, Yu

2015-01-01

Abstract Flow cytometry (FCM) is a fluorescence‐based single‐cell experimental technology that is routinely applied in biomedical research for identifying cellular biomarkers of normal physiological responses and abnormal disease states. While many computational methods have been developed that focus on identifying cell populations in individual FCM samples, very few have addressed how the identified cell populations can be matched across samples for comparative analysis. This article presents FlowMap‐FR, a novel method for cell population mapping across FCM samples. FlowMap‐FR is based on the Friedman–Rafsky nonparametric test statistic (FR statistic), which quantifies the equivalence of multivariate distributions. As applied to FCM data by FlowMap‐FR, the FR statistic objectively quantifies the similarity between cell populations based on the shapes, sizes, and positions of fluorescence data distributions in the multidimensional feature space. To test and evaluate the performance of FlowMap‐FR, we simulated the kinds of biological and technical sample variations that are commonly observed in FCM data. The results show that FlowMap‐FR is able to effectively identify equivalent cell populations between samples under scenarios of proportion differences and modest position shifts. As a statistical test, FlowMap‐FR can be used to determine whether the expression of a cellular marker is statistically different between two cell populations, suggesting candidates for new cellular phenotypes by providing an objective statistical measure. In addition, FlowMap‐FR can indicate situations in which inappropriate splitting or merging of cell populations has occurred during gating procedures. We compared the FR statistic with the symmetric version of Kullback–Leibler divergence measure used in a previous population matching method with both simulated and real data. The FR statistic outperforms the symmetric version of KL‐distance in distinguishing equivalent from nonequivalent cell populations. FlowMap‐FR was also employed as a distance metric to match cell populations delineated by manual gating across 30 FCM samples from a benchmark FlowCAP data set. An F‐measure of 0.88 was obtained, indicating high precision and recall of the FR‐based population matching results. FlowMap‐FR has been implemented as a standalone R/Bioconductor package so that it can be easily incorporated into current FCM data analytical workflows. © 2015 International Society for Advancement of Cytometry PMID:26274018

Mapping cell populations in flow cytometry data for cross-sample comparison using the Friedman-Rafsky test statistic as a distance measure.

PubMed

Hsiao, Chiaowen; Liu, Mengya; Stanton, Rick; McGee, Monnie; Qian, Yu; Scheuermann, Richard H

2016-01-01

Flow cytometry (FCM) is a fluorescence-based single-cell experimental technology that is routinely applied in biomedical research for identifying cellular biomarkers of normal physiological responses and abnormal disease states. While many computational methods have been developed that focus on identifying cell populations in individual FCM samples, very few have addressed how the identified cell populations can be matched across samples for comparative analysis. This article presents FlowMap-FR, a novel method for cell population mapping across FCM samples. FlowMap-FR is based on the Friedman-Rafsky nonparametric test statistic (FR statistic), which quantifies the equivalence of multivariate distributions. As applied to FCM data by FlowMap-FR, the FR statistic objectively quantifies the similarity between cell populations based on the shapes, sizes, and positions of fluorescence data distributions in the multidimensional feature space. To test and evaluate the performance of FlowMap-FR, we simulated the kinds of biological and technical sample variations that are commonly observed in FCM data. The results show that FlowMap-FR is able to effectively identify equivalent cell populations between samples under scenarios of proportion differences and modest position shifts. As a statistical test, FlowMap-FR can be used to determine whether the expression of a cellular marker is statistically different between two cell populations, suggesting candidates for new cellular phenotypes by providing an objective statistical measure. In addition, FlowMap-FR can indicate situations in which inappropriate splitting or merging of cell populations has occurred during gating procedures. We compared the FR statistic with the symmetric version of Kullback-Leibler divergence measure used in a previous population matching method with both simulated and real data. The FR statistic outperforms the symmetric version of KL-distance in distinguishing equivalent from nonequivalent cell populations. FlowMap-FR was also employed as a distance metric to match cell populations delineated by manual gating across 30 FCM samples from a benchmark FlowCAP data set. An F-measure of 0.88 was obtained, indicating high precision and recall of the FR-based population matching results. FlowMap-FR has been implemented as a standalone R/Bioconductor package so that it can be easily incorporated into current FCM data analytical workflows. © The Authors. Published by Wiley Periodicals, Inc. on behalf of ISAC.

Automation of sample preparation for mass cytometry barcoding in support of clinical research: protocol optimization.

PubMed

Nassar, Ala F; Wisnewski, Adam V; Raddassi, Khadir

2017-03-01

Analysis of multiplexed assays is highly important for clinical diagnostics and other analytical applications. Mass cytometry enables multi-dimensional, single-cell analysis of cell type and state. In mass cytometry, the rare earth metals used as reporters on antibodies allow determination of marker expression in individual cells. Barcode-based bioassays for CyTOF are able to encode and decode for different experimental conditions or samples within the same experiment, facilitating progress in producing straightforward and consistent results. Herein, an integrated protocol for automated sample preparation for barcoding used in conjunction with mass cytometry for clinical bioanalysis samples is described; we offer results of our work with barcoding protocol optimization. In addition, we present some points to be considered in order to minimize the variability of quantitative mass cytometry measurements. For example, we discuss the importance of having multiple populations during titration of the antibodies and effect of storage and shipping of labelled samples on the stability of staining for purposes of CyTOF analysis. Data quality is not affected when labelled samples are stored either frozen or at 4 °C and used within 10 days; we observed that cell loss is greater if cells are washed with deionized water prior to shipment or are shipped in lower concentration. Once the labelled samples for CyTOF are suspended in deionized water, the analysis should be performed expeditiously, preferably within the first hour. Damage can be minimized if the cells are resuspended in phosphate-buffered saline (PBS) rather than deionized water while waiting for data acquisition.

Modulated Raman spectroscopy for enhanced identification of bladder tumor cells in urine samples.

PubMed

Canetta, Elisabetta; Mazilu, Michael; De Luca, Anna Chiara; Carruthers, Antonia E; Dholakia, Kishan; Neilson, Sam; Sargeant, Harry; Briscoe, Tina; Herrington, C Simon; Riches, Andrew C

2011-03-01

Standard Raman spectroscopy (SRS) is a noninvasive technique that is used in the biomedical field to discriminate between normal and cancer cells. However, the presence of a strong fluorescence background detracts from the use of SRS in real-time clinical applications. Recently, we have reported a novel modulated Raman spectroscopy (MRS) technique to extract the Raman spectra from the background. In this paper, we present the first application of MRS to the identification of human urothelial cells (SV-HUC-1) and bladder cancer cells (MGH) in urine samples. These results are compared to those obtained by SRS. Classification using the principal component analysis clearly shows that MRS allows discrimination between Raman spectra of SV-HUC-1 and MGH cells with high sensitivity (98%) and specificity (95%). MRS is also used to distinguish between SV-HUC-1 and MGH cells after exposure to urine for up to 6 h. We observe a marked change in the MRS of SV-HUC-1 and MGH cells with time in urine, indicating that the conditions of sample collection will be important for the application of this methodology to clinical urine samples.

Measuring and Modeling Xenon Uptake in Plastic Beta-Cells

NASA Astrophysics Data System (ADS)

Suarez, R.; Hayes, J. C.; Harper, W. W.; Humble, P.; Ripplinger, M. D.; Stephenson, D. E.; Williams, R. M.

2013-12-01

The precision of the stable xenon volume measurement in atmospheric monitoring radio-xenon systems is a critical parameter used to determine the activity concentration of a radio-xenon sample. Typically these types of systems use a plastic scintillating beta-cell as part of a beta-gamma detection scheme to measure the radioactivity present in the gas sample. Challenges arise when performing the stable xenon calculation during or after radioactive counting of the sample due to xenon uptake into the plastic beta-cells. Plastic beta cells can adsorb as much as 5% of the sample during counting. If quantification is performed after counting, the uptake of xenon into the plastic results in an underestimation of the xenon volume measurement. This behavior also causes what is typically known as 'memory effect' in the cell. Experiments were conducted using a small volume low pressure range thermal conductivity sensor to quantify the amount of xenon uptake into the cell over a given period of time. Understanding the xenon uptake in the cell provides a better estimate of the stable volume which improves the overall measurement capability of the system. The results from these experiments along with modeling will be presented.

Internal calibration on adjacent samples (InCAS) with Fourier transform mass spectrometry.

PubMed

O'Connor, P B; Costello, C E

2000-12-15

Using matrix-assisted laser desorption/ionization (MAL DI) on a trapped ion mass spectrometer such as a Fourier transform mass spectrometer (FTMS) allows accumulation of ions in the cell from multiple laser shots prior to detection. If ions from separate MALDI samples are accumulated simultaneously in the cell, ions from one sample can be used to calibrate ions from the other sample. Since the ions are detected simultaneously in the cell, this is, in effect, internal calibration, but there are no selective desorption effects in the MALDI source. This method of internal calibration with adjacent samples is demonstrated here on cesium iodide clusters, peptides, oligosaccharides, poly(propylene glycol), and fullerenes and provides typical FTMS internal calibration mass accuracy of < 1 ppm.

Urine Cytology

MedlinePlus

... types of cells were found in your urine sample. You may need to repeat the test. Negative. This means no cancer cells were identified in your urine sample. Atypical. This indicates that some abnormalities were found ...

Low tumour cell content in a lung tumour bank: implications for molecular characterisation.

PubMed

Goh, Felicia; Duhig, Edwina E; Clarke, Belinda E; McCaul, Elizabeth; Passmore, Linda; Courtney, Deborah; Windsor, Morgan; Naidoo, Rishendren; Franz, Louise; Parsonson, Kylie; Yang, Ian A; Bowman, Rayleen V; Fong, Kwun M

2017-10-01

Lung cancer encompasses multiple malignant epithelial tumour types, each with specific targetable, potentially actionable mutations, such that precision management mandates accurate tumour typing. Molecular characterisation studies require high tumour cell content and low necrosis content, yet lung cancers are frequently a heterogeneous mixture of tumour and stromal cells. We hypothesised that there may be systematic differences in tumour cell content according to histological subtype, and that this may have implications for tumour banks as a resource for comprehensive molecular characterisation studies in lung cancer. To investigate this, we estimated tumour cell and necrosis content of 4267 samples resected from 752 primary lung tumour specimens contributed to a lung tissue bank. We found that banked lung cancer samples had low tumour cell content (33%) generally, although it was higher in carcinoids (77.5%) than other lung cancer subtypes. Tumour cells comprise a variable and often small component of banked resected tumour samples, and are accompanied by stromal reaction, inflammation, fibrosis, and normal structures. This has implications for the adequacy of unselected tumour bank samples for diagnostic and molecular investigations, and further research is needed to determine whether tumour cell content has a significant impact on analytical results in studies using tissue from tumour bank resources. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Trapping behavior of Shockley-Read-Hall recombination centers in silicon solar cells

NASA Astrophysics Data System (ADS)

Gogolin, R.; Harder, N. P.

2013-08-01

We investigate the correlation between increased apparent carrier lifetime in photoconductance-based lifetime measurements and actually reduced recombination lifetime as measured by photoluminescence measurements. These findings are further reconfirmed by I-V curve measurements of solar cells. In particular, we show experimental results for lifetime samples and solar cells with and without hydrogen passivation. In the samples and solar cells without hydrogen passivation, we find both a stronger trapping behavior and a lower recombination lifetime. Our model provides a consistent description of the observation of both, the increased apparent lifetime from carrier trapping and the decreasing recombination lifetime. In our model, both are caused by a single physical mechanism; i.e., by Recombination-Active-Trap (RAT) states. Upon fitting the experimental lifetime data, we find that the RAT-defect parameters for the hydrogen-passivated and non-hydrogen-passivated lifetime samples and solar cells are identical except for the defect concentration: hydrogen-passivation reduced the defect density by 50% in both, the lifetime samples and solar cells. We conclude that trapping should be considered as an indication for hidden, yet potentially strongly increased, low injection recombination activity.

Microfluidics cell sample preparation for analysis: Advances in efficient cell enrichment and precise single cell capture

PubMed Central

Bian, Shengtai; Cheng, Yinuo; Shi, Guanya; Liu, Peng; Ye, Xiongying

2017-01-01

Single cell analysis has received increasing attention recently in both academia and clinics, and there is an urgent need for effective upstream cell sample preparation. Two extremely challenging tasks in cell sample preparation—high-efficiency cell enrichment and precise single cell capture—have now entered into an era full of exciting technological advances, which are mostly enabled by microfluidics. In this review, we summarize the category of technologies that provide new solutions and creative insights into the two tasks of cell manipulation, with a focus on the latest development in the recent five years by highlighting the representative works. By doing so, we aim both to outline the framework and to showcase example applications of each task. In most cases for cell enrichment, we take circulating tumor cells (CTCs) as the target cells because of their research and clinical importance in cancer. For single cell capture, we review related technologies for many kinds of target cells because the technologies are supposed to be more universal to all cells rather than CTCs. Most of the mentioned technologies can be used for both cell enrichment and precise single cell capture. Each technology has its own advantages and specific challenges, which provide opportunities for researchers in their own area. Overall, these technologies have shown great promise and now evolve into real clinical applications. PMID:28217240

Size-based cell sorting with a resistive pulse sensor and an electromagnetic pump in a microfluidic chip.

PubMed

Song, Yongxin; Li, Mengqi; Pan, Xinxiang; Wang, Qi; Li, Dongqing

2015-02-01

An electrokinetic microfluidic chip is developed to detect and sort target cells by size from human blood samples. Target-cell detection is achieved by a differential resistive pulse sensor (RPS) based on the size difference between the target cell and other cells. Once a target cell is detected, the detected RPS signal will automatically actuate an electromagnetic pump built in a microchannel to push the target cell into a collecting channel. This method was applied to automatically detect and sort A549 cells and T-lymphocytes from a peripheral fingertip blood sample. The viability of A549 cells sorted in the collecting well was verified by Hoechst33342 and propidium iodide staining. The results show that as many as 100 target cells per minute can be sorted out from the sample solution and thus is particularly suitable for sorting very rare target cells, such as circulating tumor cells. The actuation of the electromagnetic valve has no influence on RPS cell detection and the consequent cell-sorting process. The viability of the collected A549 cell is not impacted by the applied electric field when the cell passes the RPS detection area. The device described in this article is simple, automatic, and label-free and has wide applications in size-based rare target cell sorting for medical diagnostics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biological response in vitro of skeletal muscle cells treated with different intensity continuous and pulsed ultrasound fields

NASA Astrophysics Data System (ADS)

Abrunhosa, Viviane M.; Mermelstein, Claudia S.; Costa, Manoel L.; Costa-Felix, Rodrigo P. B.

2011-02-01

Therapeutic ultrasound has been used in physiotherapy to accelerate tissue healing. Although the ultrasonic wave is widely used in clinical practice, not much is known about the biological effects of ultrasound on cells and tissues. This study aims to evaluate the biological response of ultrasound in primary cultures of chick myogenic cells. To ensure the metrological reliability of whole measurement process, the ultrasound equipment was calibrated in accordance with IEC 61689:2007. The skeletal muscle cells were divided in four samples. One sample was used as a control group and the others were submitted to different time and intensity and operation mode of ultrasound: 1) 0.5 W/cm2 continuous for 5 minutes, 2) 0.5 W/cm2 pulsed for 5 minutes, 3) 1.0 W/cm2 pulsed for 10 minutes. The samples were analyzed with phase contrast optical microscopy before and after the treatment. The results showed alignment of myogenic cells in the sample treated with 0.5 W/cm2 continuous during 5 minutes when compared with the control group and the other samples. This study is a first step towards a metrological and scientific based protocol to cells and tissues treatment under different ultrasound field exposures.

Interrogating Bronchoalveolar Lavage Samples via Exclusion-Based Analyte Extraction.

PubMed

Tokar, Jacob J; Warrick, Jay W; Guckenberger, David J; Sperger, Jamie M; Lang, Joshua M; Ferguson, J Scott; Beebe, David J

2017-06-01

Although average survival rates for lung cancer have improved, earlier and better diagnosis remains a priority. One promising approach to assisting earlier and safer diagnosis of lung lesions is bronchoalveolar lavage (BAL), which provides a sample of lung tissue as well as proteins and immune cells from the vicinity of the lesion, yet diagnostic sensitivity remains a challenge. Reproducible isolation of lung epithelia and multianalyte extraction have the potential to improve diagnostic sensitivity and provide new information for developing personalized therapeutic approaches. We present the use of a recently developed exclusion-based, solid-phase-extraction technique called SLIDE (Sliding Lid for Immobilized Droplet Extraction) to facilitate analysis of BAL samples. We developed a SLIDE protocol for lung epithelial cell extraction and biomarker staining of patient BALs, testing both EpCAM and Trop2 as capture antigens. We characterized captured cells using TTF1 and p40 as immunostaining biomarkers of adenocarcinoma and squamous cell carcinoma, respectively. We achieved up to 90% (EpCAM) and 84% (Trop2) extraction efficiency of representative tumor cell lines. We then used the platform to process two patient BAL samples in parallel within the same sample plate to demonstrate feasibility and observed that Trop2-based extraction potentially extracts more target cells than EpCAM-based extraction.

Ultrasonic standing wave preparation of a liquid cell for glucose measurements in urine by midinfrared spectroscopy and potential application to smart toilets.

PubMed

Yamamoto, Naoyuki; Kawashima, Natsumi; Kitazaki, Tomoya; Mori, Keita; Kang, Hanyue; Nishiyama, Akira; Wada, Kenji; Ishimaru, Ichiro

2018-05-01

Smart toilets could be used to monitor different components of urine in daily life for early detection of lifestyle-related diseases and prompt provision of treatment. For analysis of biological samples such as urine by midinfrared spectroscopy, thin-film samples like liquid cells are needed because of the strong absorption of midinfrared light by water. Conventional liquid cells or fixed cells are prepared based on the liquid membrane method and solution technique, but these are not quantitative and are difficult to set up and clean. We generated an ultrasonic standing wave reflection plane in a sample and produced an ultrasonic liquid cell. In this cell, the thickness of the optical path length was adjustable, as in the conventional method. The reflection plane could be generated at an arbitrary depth and internal reflected light could be detected by changing the frequency of the ultrasonic wave. We could generate refractive index boundaries using the density difference created by the ultrasonic standing wave. Creation of the reflection plane in the sample was confirmed by optical coherence tomography. Using the proposed method and midinfrared spectroscopy, we discriminated between normal urine samples spiked with glucose at different concentrations and obtained a high correlation coefficient. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Simultaneous extraction of proteins and metabolites from cells in culture

PubMed Central

Sapcariu, Sean C.; Kanashova, Tamara; Weindl, Daniel; Ghelfi, Jenny; Dittmar, Gunnar; Hiller, Karsten

2014-01-01

Proper sample preparation is an integral part of all omics approaches, and can drastically impact the results of a wide number of analyses. As metabolomics and proteomics research approaches often yield complementary information, it is desirable to have a sample preparation procedure which can yield information for both types of analyses from the same cell population. This protocol explains a method for the separation and isolation of metabolites and proteins from the same biological sample, in order for downstream use in metabolomics and proteomics analyses simultaneously. In this way, two different levels of biological regulation can be studied in a single sample, minimizing the variance that would result from multiple experiments. This protocol can be used with both adherent and suspension cell cultures, and the extraction of metabolites from cellular medium is also detailed, so that cellular uptake and secretion of metabolites can be quantified. Advantages of this technique includes:1.Inexpensive and quick to perform; this method does not require any kits.2.Can be used on any cells in culture, including cell lines and primary cells extracted from living organisms.3.A wide variety of different analysis techniques can be used, adding additional value to metabolomics data analyzed from a sample; this is of high value in experimental systems biology. PMID:26150938

Toxicity bioassays with concentrated cell culture media-a methodology to overcome the chemical loss by conventional preparation of water samples.

PubMed

Niss, Frida; Rosenmai, Anna Kjerstine; Mandava, Geeta; Örn, Stefan; Oskarsson, Agneta; Lundqvist, Johan

2018-04-01

The use of in vitro bioassays for studies of toxic activity in environmental water samples is a rapidly expanding field of research. Cell-based bioassays can assess the total toxicity exerted by a water sample, regardless whether the toxicity is caused by a known or unknown agent or by a complex mixture of different agents. When using bioassays for environmental water samples, it is often necessary to concentrate the water samples before applying the sample. Commonly, water samples are concentrated 10-50 times. However, there is always a risk of losing compounds in the sample in such sample preparation. We have developed an alternative experimental design by preparing a concentrated cell culture medium which was then diluted in the environmental water sample to compose the final cell culture media for the in vitro assays. Water samples from five Swedish waste water treatment plants were analyzed for oxidative stress response, estrogen receptor (ER), and aryl hydrocarbon receptor (AhR) activity using this experimental design. We were able to detect responses equivalent to 8.8-11.3 ng/L TCCD for AhR activity and 0.4-0.9 ng/L 17β-estradiol for ER activity. We were unable to detect oxidative stress response in any of the studied water samples. In conclusion, we have developed an experimental design allowing us to examine environmental water samples in toxicity in vitro assays at a concentration factor close to 1, without the risk of losing known or unknown compounds during an extraction procedure.

The examination of urine samples for pathogenic microbes by the luciferase assay for ATP. 1: The effect of the presence of fungi, fungal like bacteria and kidney cells in urine samples

NASA Technical Reports Server (NTRS)

Bush, V. N.

1973-01-01

A method for accurately determining urinary tract infections in man is introduced. The method is based on adenosine triphosphate (ATP) concentration in urine samples after removing nonbacterial ATP. Adenosine triphosphate concentration is measured from the bioluminescent reaction of luciferase when mixed with ATP. An examination was also made of the effectiveness of rupturing agents on monkey kidney cells Candia albicans, a Rhodotorula species, and a Streptomyces species in determining whether these cells could contribute ATP to the bacterial ATP value of a urine sample.

Questioning the utility of pooling samples in microarray experiments with cell lines.

PubMed

Lusa, L; Cappelletti, V; Gariboldi, M; Ferrario, C; De Cecco, L; Reid, J F; Toffanin, S; Gallus, G; McShane, L M; Daidone, M G; Pierotti, M A

2006-01-01

We describe a microarray experiment using the MCF-7 breast cancer cell line in two different experimental conditions for which the same number of independent pools as the number of individual samples was hybridized on Affymetrix GeneChips. Unexpectedly, when using individual samples, the number of probe sets found to be differentially expressed between treated and untreated cells was about three times greater than that found using pools. These findings indicate that pooling samples in microarray experiments where the biological variability is expected to be small might not be helpful and could even decrease one's ability to identify differentially expressed genes.

Evaluation of different continuous cell lines in the isolation of mumps virus by the shell vial method from clinical samples

PubMed Central

Reina, J; Ballesteros, F; Mari, M; Munar, M

2001-01-01

Aims—To compare prospectively the efficacy of the Vero, LLC-MK2, MDCK, Hep-2, and MRC-5 cell lines in the isolation of the mumps virus from clinical samples by means of the shell vial method. Methods—During an epidemic outbreak of parotiditis 48 clinical samples (saliva swabs and CSF) were studied. Two vials of the Vero, LLC-MK2, MDCK, MRC-5, and Hep-2 cell lines were inoculated with 0.2 ml of the samples by the shell vial assay. The vials were incubated at 36°C for two and five days. The vials were then fixed with acetone at -20°C for 10 minutes and stained by a monoclonal antibody against mumps virus by means of an indirect immunofluorescence assay. Results—The mumps virus was isolated from 36 samples. The Vero and LLC-MK2 cell lines showed a 100% isolation capacity, MDCK showed 77.7%, MRC-5 showed 44.4%, and Hep-2 showed 22.2%. The Vero and LLC-MK2 lines were significantly different to the other cell lines (p < 0.001). The sensitivity for the Vero and LLC-MK2 lines at two and five days of incubation was identical (100%). The values obtained in the study of the quantitative isolation capacity (positive isolation with > 5 infectious foci) were 94.4% for Vero, 97.2% for LLC-MK2, 5.5% for MDCK, 5.5% for Hep-2, and 0% for MRC-5. Conclusions—The Vero and LLC-MK2 cell lines are equally efficient at two and five days incubation for the isolation of the mumps virus from clinical samples, and the use of the shell vial method considerably shortens the time of aetiological diagnosis with higher specificity. Key Words: mumps virus • Vero cell line • LLC-MK2 cell line • MDCK cell line • Hep-2 cell line • MRC-5 cell line • isolation • shell vial PMID:11729211

Effect of Water-Glass Coating on HA and HA-TCP Samples for MSCs Adhesion, Proliferation, and Differentiation.

PubMed

Bajpai, Indu; Kim, Duk Yeon; Kyong-Jin, Jung; Song, In-Hwan; Kim, Sukyoung

2016-01-01

Ca-P and silicon based materials have become very popular as bone tissue engineering materials. In this study, water-glass (also known as sodium silicate glass) was coated on sintered hydroxyapatite (HA) and HA-TCP (TCP stands for tricalcium phosphate) samples and subsequently heat-treated at 600°C for 2 hrs. X-rays diffraction showed the presence of β- and α-TCP phases along with HA in the HA-TCP samples. Samples without coating, with water-glass coating, and heat-treated after water-glass coating were used to observe the adhesion and proliferation response of bone marrow derived-mesenchymal stem cells (MSCs). Cell culture was carried out for 4 hrs, 1 day, and 7 days. Interestingly, all samples showed similar response for cell adhesion and proliferation up to 7-day culture but fibronectin, E-cadherin, and osteogenic differentiation related genes (osteocalcin and osteopontin) were significantly induced in heat-treated water-glass coated HA-TCP samples. A water-glass coating on Ca-P samples was not found to influence the cell proliferation response significantly but activated some extracellular matrix genes and induced osteogenic differentiation in the MSCs.

Osteoclastogenesis and Osteoclastic Resorption of Tricalcium Phosphate: Effect of Strontium and Magnesium Doping

PubMed Central

Roy, Mangal; Bose, Susmita

2012-01-01

Bone substitute materials are required to support the remodeling process, which consists of osteoclastic resorption and osteoblastic synthesis. Osteoclasts, the bone resorbing cells, generate from differentiation of hemopoietic mononuclear cells. In the present study we have evaluated the effects of 1.0 wt% strontium (Sr) and 1.0 wt% magnesium (Mg) doping in beta-tricalcium phosphate (β-TCP) on the differentiation of mononuclear cells into osteoclast-like cells and its resorptive activity. In vitro osteoclast-like cell formation, adhesion, and resorption were studied using osteoclast precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Osteoclast-like cell formation was noticed on pure and Sr doped β-TCP samples at day 8 which was absent on Mg doped β-TCP samples indicating decrease in initial osteoclast differentiation due to Mg doping. After 21 days of culture, osteoclast-like cell formation was evident on all samples with osteoclastic markers such as actin ring, multiple nuclei, and presence of vitronectin receptor αvβ3 integrin. After osteoclast differentiation, all substrates showed osteoclast-like cell mediated degradation, however; significantly restricted for Mg doped β-TCP samples. Our present results indicated substrate chemistry controlled osteoclast differentiation and resorptive activity which can be used in designing TCP based resorbable bone substitutes with controlled degradation properties. PMID:22566212

Osteoclastogenesis and osteoclastic resorption of tricalcium phosphate: effect of strontium and magnesium doping.

PubMed

Roy, Mangal; Bose, Susmita

2012-09-01

Bone substitute materials are required to support the remodeling process, which consists of osteoclastic resorption and osteoblastic synthesis. Osteoclasts, the bone-resorbing cells, generate from differentiation of hemopoietic mononuclear cells. In the present study, we have evaluated the effects of 1.0 wt % strontium (Sr) and 1.0 wt % magnesium (Mg) doping in beta-tricalcium phosphate (β-TCP) on the differentiation of mononuclear cells into osteoclast-like cells and its resorptive activity. In vitro osteoclast-like cell formation, adhesion, and resorption were studied using osteoclast precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Osteoclast-like cell formation was noticed on pure and Sr-doped β-TCP samples at day 8, which was absent on Mg-doped β-TCP samples indicating decrease in initial osteoclast differentiation due to Mg doping. After 21 days of culture, osteoclast-like cell formation was evident on all samples with osteoclastic markers such as actin ring, multiple nuclei, and presence of vitronectin receptor α(v)β(3) integrin. After osteoclast differentiation, all substrates showed osteoclast-like cell-mediated degradation, however, significantly restricted for Mg-doped β-TCP samples. Our present results indicated that substrate chemistry controlled osteoclast differentiation and resorptive activity, which can be used in designing TCP-based resorbable bone substitutes with controlled degradation properties. Copyright © 2012 Wiley Periodicals, Inc.

A cell extraction method for oily sediments

NASA Astrophysics Data System (ADS)

Lappé, M.; Kallmeyer, J.

2012-04-01

Hydrocarbons can be found in many different habitats and represent an important carbon source for microbes. As fossil fuels, they are an important economical resource and, through natural seepage or accidental release, they can be major pollutants. Oil sands from Alberta, Canada, and samples from the seafloor of the Gulf of Mexico represent typical examples of either natural or anthropogenically affected oily sediments. DNA-specific stains and molecular probes bind to hydrocarbons, causing massive background fluorescence and thereby massively hampering cell enumeration. The cell extraction procedure of Kallmeyer et al. (2008) separates the cells from the sediment matrix, producing a sediment free cell extract that can then be used for subsequent staining and cell enumeration under a fluorescence microscope. In principle, this technique can also be used to separate cells from oily sediments, but it was not originally optimized for this application and does not provide satisfactory results. Here we present a modified extraction method in which the hydrocarbons are removed prior to cell extraction by a solvent treatment. Due to the reduced background fluorescence the microscopic image becomes clearer, making cell identification and enumeration much easier. Consequently, the resulting cell counts from oily samples treated according to our new protocol were significantly higher than those treated according to Kallmeyer et al. (2008). We tested different amounts of a variety of solvents for their ability to remove hydrocarbons and found that n-hexane and - in samples containing more biodegraded oils - methanol, delivered the best results. Because solvents also tend to lyse cells, it was important to find the optimum solvent to sample ratio, at which the positive effect of hydrocarbon extraction overcomes the negative effect of cell lysis. A volumetric ratio of 1:2 to 1:5 between a formalin-fixed sediment slurry and solvent delivered highest cell counts. Extraction efficiency was around 30 to 50% and was checked on both oily samples spiked with known amounts of E.coli cells and oil-free samples amended with non-biodegraded and biodegraded oil. The method provided reproducible results on samples containing very different kinds of oils with regard to their degree of biodegradation. For strongly biodegraded oils, like those from the Alberta oil sands, methanol turned out to be the most appropriate solvent. For less biodegraded oils, like those from sediments from the Gulf of Mexico, n-hexane delivered best results. The relative amount of polar groups increases with an increasing level of biodegradation. Therefore polar solvents like methanol are better suited to dissolve biodegraded oils than less polar solvents like n-hexane. Our new method only provides a minimum estimate of cell abundance, as some cells are either lysed by the solvent treatment or remain attached to mineral grains and therefore do not end up in the cell extract but remain in the sediment pellet which will not be used for further microbiological analysis.

Population transcriptomics with single-cell resolution: a new field made possible by microfluidics: a technology for high throughput transcript counting and data-driven definition of cell types.

PubMed

Plessy, Charles; Desbois, Linda; Fujii, Teruo; Carninci, Piero

2013-02-01

Tissues contain complex populations of cells. Like countries, which are comprised of mixed populations of people, tissues are not homogeneous. Gene expression studies that analyze entire populations of cells from tissues as a mixture are blind to this diversity. Thus, critical information is lost when studying samples rich in specialized but diverse cells such as tumors, iPS colonies, or brain tissue. High throughput methods are needed to address, model and understand the constitutive and stochastic differences between individual cells. Here, we describe microfluidics technologies that utilize a combination of molecular biology and miniaturized labs on chips to study gene expression at the single cell level. We discuss how the characterization of the transcriptome of each cell in a sample will open a new field in gene expression analysis, population transcriptomics, that will change the academic and biomedical analysis of complex samples by defining them as quantified populations of single cells. Copyright © 2013 WILEY Periodicals, Inc.

Digital Holographic Microscopy, a Method for Detection of Microorganisms in Plume Samples from Enceladus and Other Icy Worlds

PubMed Central

Bedrossian, Manuel; Lindensmith, Chris

2017-01-01

Abstract Detection of extant microbial life on Earth and elsewhere in the Solar System requires the ability to identify and enumerate micrometer-scale, essentially featureless cells. On Earth, bacteria are usually enumerated by culture plating or epifluorescence microscopy. Culture plates require long incubation times and can only count culturable strains, and epifluorescence microscopy requires extensive staining and concentration of the sample and instrumentation that is not readily miniaturized for space. Digital holographic microscopy (DHM) represents an alternative technique with no moving parts and higher throughput than traditional microscopy, making it potentially useful in space for detection of extant microorganisms provided that sufficient numbers of cells can be collected. Because sample collection is expected to be the limiting factor for space missions, especially to outer planets, it is important to quantify the limits of detection of any proposed technique for extant life detection. Here we use both laboratory and field samples to measure the limits of detection of an off-axis digital holographic microscope (DHM). A statistical model is used to estimate any instrument's probability of detection at various bacterial concentrations based on the optical performance characteristics of the instrument, as well as estimate the confidence interval of detection. This statistical model agrees well with the limit of detection of 103 cells/mL that was found experimentally with laboratory samples. In environmental samples, active cells were immediately evident at concentrations of 104 cells/mL. Published estimates of cell densities for Enceladus plumes yield up to 104 cells/mL, which are well within the off-axis DHM's limits of detection to confidence intervals greater than or equal to 95%, assuming sufficient sample volumes can be collected. The quantitative phase imaging provided by DHM allowed minerals to be distinguished from cells. Off-axis DHM's ability for rapid low-level bacterial detection and counting shows its viability as a technique for detection of extant microbial life provided that the cells can be captured intact and delivered to the sample chamber in a sufficient volume of liquid for imaging. Key Words: In situ life detection—Extant microorganisms—Holographic microscopy—Ocean Worlds—Enceladus—Imaging. Astrobiology 17, 913–925. PMID:28708412

Microsystem strategies for sample preparation in biological detection.

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, Conrad D.; Galambos, Paul C.; Bennett, Dawn Jonita

2005-03-01

The objective of this LDRD was to develop microdevice strategies for dealing with samples to be examined in biological detection systems. This includes three sub-components: namely, microdevice fabrication, sample delivery to the microdevice, and sample processing within the microdevice. The first component of this work focused on utilizing Sandia's surface micromachining technology to fabricate small volume (nanoliter) fluidic systems for processing small quantities of biological samples. The next component was to develop interfaces for the surface-micromachined silicon devices. We partnered with Micronics, a commercial company, to produce fluidic manifolds for sample delivery to our silicon devices. Pressure testing was completedmore » to examine the strength of the bond between the pressure-sensitive adhesive layer and the silicon chip. We are also pursuing several other methods, both in house and external, to develop polymer-based fluidic manifolds for packaging silicon-based microfluidic devices. The second component, sample processing, is divided into two sub-tasks: cell collection and cell lysis. Cell collection was achieved using dielectrophoresis, which employs AC fields to collect cells at energized microelectrodes, while rejecting non-cellular particles. Both live and dead Staph. aureus bacteria have been collected using RF frequency dielectrophoresis. Bacteria have been separated from polystyrene microspheres using frequency-shifting dielectrophoresis. Computational modeling was performed to optimize device separation performance, and to predict particle response to the dielectrophoretic traps. Cell lysis is continuing to be pursued using microactuators to mechanically disrupt cell membranes. Novel thermal actuators, which can generate larger forces than previously tested electrostatic actuators, have been incorporated with and tested with cell lysis devices. Significant cell membrane distortion has been observed, but more experiments need to be conducted to determine the effects of the observed distortion on membrane integrity and cell viability. Finally, we are using a commercial PCR DNA amplification system to determine the limits of detectable sample size, and to examine the amplification of DNA bound to microspheres. Our objective is to use microspheres as capture-and-carry chaperones for small molecules such as DNA and proteins, enabling the capture and concentration of the small molecules using dielectrophoresis. Current tests demonstrated amplification of DNA bound to micron-sized polystyrene microspheres using 20-50 microliter volume size reactions.« less

DALMATIAN: An Algorithm for Automatic Cell Detection and Counting in 3D.

PubMed

Shuvaev, Sergey A; Lazutkin, Alexander A; Kedrov, Alexander V; Anokhin, Konstantin V; Enikolopov, Grigori N; Koulakov, Alexei A

2017-01-01

Current 3D imaging methods, including optical projection tomography, light-sheet microscopy, block-face imaging, and serial two photon tomography enable visualization of large samples of biological tissue. Large volumes of data obtained at high resolution require development of automatic image processing techniques, such as algorithms for automatic cell detection or, more generally, point-like object detection. Current approaches to automated cell detection suffer from difficulties originating from detection of particular cell types, cell populations of different brightness, non-uniformly stained, and overlapping cells. In this study, we present a set of algorithms for robust automatic cell detection in 3D. Our algorithms are suitable for, but not limited to, whole brain regions and individual brain sections. We used watershed procedure to split regional maxima representing overlapping cells. We developed a bootstrap Gaussian fit procedure to evaluate the statistical significance of detected cells. We compared cell detection quality of our algorithm and other software using 42 samples, representing 6 staining and imaging techniques. The results provided by our algorithm matched manual expert quantification with signal-to-noise dependent confidence, including samples with cells of different brightness, non-uniformly stained, and overlapping cells for whole brain regions and individual tissue sections. Our algorithm provided the best cell detection quality among tested free and commercial software.

Performance Enhancement of 3-Mercaptopropionic Acid-Capped CdSe Quantum-Dot Sensitized Solar Cells Incorporating Single-Walled Carbon Nanotubes.

PubMed

Yang, Jonghee; Park, Taehee; Lee, Jongtaek; Lee, Junyoung; Shin, Hokyeong; Yi, Whikun

2016-03-01

We fabricated a series of linker-assisted quantum-dot-sensitized solar cells based on the ex situ self-assembly of CdSe quantum dots (QDs) onto TiO2 electrode using sulfide/polysulfide (S(2-)/Sn(2-)) as an electrolyte and Au cathode. Our cell were combined with single-walled carbon nanotubes (SWNTs) by two techniques; One was mixing SWNTs with TiO2 electrode and the other was spraying SWNTs onto Au electrode. Absorption spectra were used to confirm the adsorption of QDs onto TiO2 electrode. Cell performance was measured on samples containing and not-containing SWNTs. Samples mixing SWNTs with TiO2 showed higher cell efficiency, on the while sample spraying SWNTs onto Au electrode showed lower efficiency compared with pristine sample (not-containing SWNTs). Electrochemical impedance spectroscopy analysis suggested that SWNTs can act as either barriers or excellent carrier transfers according their position and mixing method.

High resolution Fourier interferometer-spectrophotopolarimeter

NASA Technical Reports Server (NTRS)

Fymat, A. L. (Inventor)

1976-01-01

A high-resolution Fourier interferometer-spectrophotopolarimeter is provided using a single linear polarizer-analyzer the transmission axis azimuth of which is positioned successively in the three orientations of 0 deg, 45 deg, and 90 deg, in front of a detector; four flat mirrors, three of which are switchable to either of two positions to direct an incoming beam from an interferometer to the polarizer-analyzer around a sample cell transmitted through a medium in a cell and reflected by medium in the cell; and four fixed focussing lenses, all located in a sample chamber attached at the exit side of the interferometer. This arrangement can provide the distribution of energy and complete polarization state across the spectrum of the reference light entering from the interferometer; the same light after a fixed-angle reflection from the sample cell containing a medium to be analyzed; and the same light after direct transmission through the same sample cell, with the spectral resolution provided by the interferometer.

Active Sampling State Dynamically Enhances Olfactory Bulb Odor Representation.

PubMed

Jordan, Rebecca; Fukunaga, Izumi; Kollo, Mihaly; Schaefer, Andreas T

2018-06-27

The olfactory bulb (OB) is the first site of synaptic odor information processing, yet a wealth of contextual and learned information has been described in its activity. To investigate the mechanistic basis of contextual modulation, we use whole-cell recordings to measure odor responses across rapid learning episodes in identified mitral/tufted cells (MTCs). Across these learning episodes, diverse response changes occur already during the first sniff cycle. Motivated mice develop active sniffing strategies across learning that robustly correspond to the odor response changes, resulting in enhanced odor representation. Evoking fast sniffing in different behavioral states demonstrates that response changes during active sampling exceed those predicted from feedforward input alone. Finally, response changes are highly correlated in tufted cells, but not mitral cells, indicating there are cell-type-specific effects on odor representation during active sampling. Altogether, we show that active sampling is strongly associated with enhanced OB responsiveness on rapid timescales. Copyright © 2018 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

Constant pressure high throughput membrane permeation testing system

DOEpatents

Albenze, Erik J.; Hopkinson, David P.; Luebke, David R.

2014-09-02

The disclosure relates to a membrane testing system for individual evaluation of a plurality of planar membranes subjected to a feed gas on one side and a sweep gas on a second side. The membrane testing system provides a pressurized flow of a feed and sweep gas to each membrane testing cell in a plurality of membrane testing cells while a stream of retentate gas from each membrane testing cell is ported by a retentate multiport valve for sampling or venting, and a stream of permeate gas from each membrane testing cell is ported by a permeate multiport valve for sampling or venting. Back pressure regulators and mass flow controllers act to maintain substantially equivalent gas pressures and flow rates on each side of the planar membrane throughout a sampling cycle. A digital controller may be utilized to position the retentate and permeate multiport valves cyclically, allowing for gas sampling of different membrane cells over an extended period of time.

3D nanoscale imaging of the yeast, Schizosaccharomyces pombe, by full-field transmission x-ray microscopy at 5.4 keV

PubMed Central

Chen, Jie; Yang, Yunhao; Zhang, Xiaobo; Andrews, Joy C.; Pianetta, Piero; Guan, Yong; Liu, Gang; Xiong, Ying; Wu, Ziyu; Tian, Yangchao

2010-01-01

Three-dimensional (3D) nanoscale structures of the fission yeast, Schizosaccharomyces pombe, can be obtained by full-field transmission hard x-ray microscopy with 30 nm resolution using synchrotron radiation sources. Sample preparation is relatively simple and the samples are portable across various imaging environments, allowing for high throughput sample screening. The yeast cells were fixed and double stained with Reynold’s lead citrate and uranyl acetate. We performed both absorption contrast and Zernike phase contrast imaging on these cells in order to test this method. The membranes, nucleus and subcellular organelles of the cells were clearly visualized using absorption contrast mode. The x-ray images of the cells could be used to study the spatial distributions of the organelles in the cells. These results show unique structural information, demonstrating that hard x-ray microscopy is a complementary method for imaging and analyzing biological samples. PMID:20349228

Preparation of pure microbiological samples for pyrolysis gas-liquid chromatography studies

NASA Technical Reports Server (NTRS)

Oxborrow, G. S.; Fields, N. D.; Puleo, J. R.

1976-01-01

Bacterial samples were prepared for pyrolysis gas-liquid chromatography using cells grown on membrane filters. Pyrochromatograms were reproducible when cells harvested from the filters were pyrolyzed without being washed.

Traceability in stem cell research: from participant sample to induced pluripotent stem cell and back.

PubMed

Morrison, Michael; Moraia, Linda Briceño; Steele, Jane C

2016-01-01

This paper describes a traceability system developed for the Stem cells for Biological Assays of Novel drugs and prediCtive toxiCology consortium. The system combines records and labels that to biological material across geographical locations and scientific processes from sample donation to induced pluripotent stem cell line. The labeling system uses a unique identification number to link every aliquot of sample at every stage of the reprogramming pathway back to the original donor. Only staff at the clinical recruitment site can reconnect the unique identification number to the identifying details of a specific donor. This ensures the system meets ethical and legal requirements for protecting privacy while allowing full traceability of biological material. The system can be adapted to other projects and for use with different primary sample types.

Immune privilege of the CNS is not the consequence of limited antigen sampling

NASA Astrophysics Data System (ADS)

Harris, Melissa G.; Hulseberg, Paul; Ling, Changying; Karman, Jozsef; Clarkson, Benjamin D.; Harding, Jeffrey S.; Zhang, Mengxue; Sandor, Adam; Christensen, Kelsey; Nagy, Andras; Sandor, Matyas; Fabry, Zsuzsanna

2014-03-01

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

Percolation in a Proton Exchange Membrane Fuel Cell Catalyst Layer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stacy, Stephen; Allen, Jeffrey

Water management in the catalyst layers of proton exchange membrane fuel cells (PEMFC) is confronted by two issues, flooding and dry out, both of which result in improper functioning of the fuel cell and lead to poor performance and degradation. At the present time, the data that has been reported about water percolation and wettability within a fuel cell catalyst layer is limited. A method and apparatus for measuring the percolation pressure in the catalyst layer has been developed based upon an experimental apparatus used to test water percolation in porous transport layers (PTL). The experimental setup uses a pseudomore » Hele-Shaw type testing where samples are compressed and a fluid is injected into the sample. Testing the samples gives percolation pressure plots which show trends in increasing percolation pressure with an increase in flow rate. A decrease in pressure was seen as percolation occurred in one sample, however the pressure only had a rising effect in the other sample.« less

Electrochemically Initiated Tagging of Thiols Using an Electrospray Ionization-Based Liquid Microjunction Surface Sampling Probe Two-Electrode Cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Berkel, Gary J; Kertesz, Vilmos

2009-01-01

This paper reports on the conversion of a liquid microjunction surface sampling probe (LMJ-SSP) into a two electrode electrochemical cell using a conductive sample surface and the probe as the two electrodes with an appropriate battery powered circuit. With this LMJ-SSP, two-electrode cell arrangement, tagging of analyte thiol functionalities (in this case peptide cysteine residues) with hydroquinone tags was initiated electrochemically using a hydroquinone doped solution when the analyte either was initially in solution or was sampled from a surface. Efficient tagging (~90%), at flow rates of 5-10 L/min, could be achieved for up to at least two cysteines onmore » a peptide. The high tagging efficiency observed was explained with a simple kinetic model. In general, the incorporation of a two-electrode electrochemical cell, or other multiple electrode arrangement, into the LMJ-SSP is expected to add to the versatility of this approach for surface sampling and ionization coupled with mass spectrometric detection.« less

Different tissue phagocytes sample apoptotic cells to direct distinct homeostasis programs.

PubMed

Cummings, Ryan J; Barbet, Gaetan; Bongers, Gerold; Hartmann, Boris M; Gettler, Kyle; Muniz, Luciana; Furtado, Glaucia C; Cho, Judy; Lira, Sergio A; Blander, J Magarian

2016-11-24

Recognition and removal of apoptotic cells by professional phagocytes, including dendritic cells and macrophages, preserves immune self-tolerance and prevents chronic inflammation and autoimmune pathologies. The diverse array of phagocytes that reside within different tissues, combined with the necessarily prompt nature of apoptotic cell clearance, makes it difficult to study this process in situ. The full spectrum of functions executed by tissue-resident phagocytes in response to homeostatic apoptosis, therefore, remains unclear. Here we show that mouse apoptotic intestinal epithelial cells (IECs), which undergo continuous renewal to maintain optimal barrier and absorptive functions, are not merely extruded to maintain homeostatic cell numbers, but are also sampled by a single subset of dendritic cells and two macrophage subsets within a well-characterized network of phagocytes in the small intestinal lamina propria. Characterization of the transcriptome within each subset before and after in situ sampling of apoptotic IECs revealed gene expression signatures unique to each phagocyte, including macrophage-specific lipid metabolism and amino acid catabolism, and a dendritic-cell-specific program of regulatory CD4 + T-cell activation. A common 'suppression of inflammation' signature was noted, although the specific genes and pathways involved varied amongst dendritic cells and macrophages, reflecting specialized functions. Apoptotic IECs were trafficked to mesenteric lymph nodes exclusively by the dendritic cell subset and served as critical determinants for the induction of tolerogenic regulatory CD4 + T-cell differentiation. Several of the genes that were differentially expressed by phagocytes bearing apoptotic IECs overlapped with susceptibility genes for inflammatory bowel disease. Collectively, these findings provide new insights into the consequences of apoptotic cell sampling, advance our understanding of how homeostasis is maintained within the mucosa and set the stage for development of novel therapeutics to alleviate chronic inflammatory diseases such as inflammatory bowel disease.

Microbial flora on cell-phones in an orthopedic surgery room before and after decontamination.

PubMed

Murgier, J; Coste, J-F; Cavaignac, E; Bayle-Iniguez, X; Chiron, P; Bonnevialle, P; Laffosse, J-M

2016-12-01

Cell-phones are the typical kind of object brought into the operating room from outside by hospital staff. A great effort is made to reduce the level of potentially contaminating bacteria in the operating room, and introducing these devices may run counter to good practice. The study hypothesis was that cell-phones are colonized by several strains of bacteria and may constitute a source of nosocomial contamination. The main study objective was to screen for bacterial colonies on the surfaces of cell-phones introduced in an orthopedic surgery room. The secondary objective was to assess the efficacy of decontamination. Samples were taken from the cell-phones of hospital staff (surgeons, anaesthetists, nurses, radiology operators, and external medical representatives) entering the operating room of the university hospital center orthopedic surgery department, Toulouse (France). Sampling used Count Tact ® contact gel, without wiping the phone down in advance. Both sides of the phone were sampled, before and after decontamination with a pad imbibed with 0.25% Surfanios ® Premium disinfectant. A nasal sample was also taken to investigate the correlation between Staphylococcus aureus in the nasal cavities and on the cell-phone. Fifty-two cell-phones were sampled. Before decontamination, the mean number of colony-forming units (CFU) was 258 per phone (range, 0-1,664). After decontamination, it was 127 (range, 0-800) (P=0.0001). Forty-nine cell-phones bore CFUs before decontamination (94%), and 39 after (75%) (P=0.02). Cell-phones are CFU carriers and may thus lead to contamination. Guidelines should be drawn up to encourage cleaning phones regularly and to reduce levels of use within the operating room. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Expression of Vascular Endothelial Growth Factor in Odontogenic Cysts: Is There Any Impression on Clinical Outcome?

PubMed

Sadri, Donia; Farhadi, Sareh; Shahabi, Zahra; Sarshar, Samaneh

2016-01-01

The recent scientific reports have shown that angiogenesis can affect biological behavior of pathologic lesions. Regarding unique clinical outcome of Odontogenic keratocyst (OKC), the present study was aimed to compare angiogenesis in Odontogenic keratocyst and Dentigerous cyst (DC). In this experimental study, tissue sections of 46 samples of OKC and DC were stained through immunohistochemical method using Vascular Endothelial Growth Factor (VEGF) antibody. VEGF expression was evaluated in epithelial cells, fibroblasts and endothelial cells. The average percentage of stained cells in any samples was categorized to 3 groups as follows: SCORE 0: 10% of cells or less are positive. SCORE 1: 10 to 50% of cells are positive. SCORE 2: more than 50% of cells are positive. Mann-U-Whitney, T-test and chi-square was used for statistical analysis. The average of VEGF expression in 24 samples of DC was 20.2% and in 22 samples of OKC was 52.6%, respectively. The average of VEGF expression in these two cysts had statistical significant differences. (PV= 0.045). There was significant statistical differences between two cysts in the terms of VEGF SCORE (PV= 0.000). OKC samples had significantly higher SCORE for the purpose of VEGF incidence than DC. Also, there were no differences between VEGF expression in epithelial cells of two cysts (PV= 0.268) there were significant statistical differences between two cysts in terms of endothelial cell staining. The endothelial cell staining was significantly higher in OKC than DC (PV= 0.037%). Regarding higher expression of Vascular Endothelial Growth factor in OKC than DC, it seems that angiogenesis may have great impression on clinical outcome of OKC.

Characterization of dendritic cells in lip and oral cavity squamous cell carcinoma.

PubMed

Costa, Nádia Lago; Gonçalves, Andréia Souza; Martins, Allisson Filipe Lopes; Arantes, Diego Antônio Costa; Silva, Tarcília Aparecida; Batista, Aline Carvalho

2016-07-01

There may be differences in the antitumor immunity induced by dendritic cells (DCs) during the development of squamous cell carcinoma (SCC) located in the lip rather than in the oral cavity. The aim of this study was to evaluate the number of immature and mature DCs in SCC and potentially malignant disorders of the oral cavity and lip. Immunohistochemistry was used to identify the number (cells/mm(2) ) of immature (CD1a(+) ) or mature (CD83(+) ) DCs in samples of oral cavity SCC (OCSCC) (n = 39), lip SCC (LSCC) (n = 23), leukoplakia (LK) (n = 21), actinic cheilitis (AC) (n = 13), and normal mucosa of the oral cavity (OC control, n = 12) and the lip (lip control, n = 11). The number of CD1a(+) cells tended to be higher in the OC control samples compared with the LK (P = 0.04) and OCSCC (P = 0.21). Unlike, this cell population was lower in the lip control than in AC or LSCC (P < 0.05). The number of CD83(+) cells was increased in the LSCC samples compared with the AC and lip control (P = 0.0001) and in OCSCC compared with both the LK (P = 0.001) and OC control (P = 0.0001) samples. LSCC showed an elevated number of CD1a(+) and CD83(+) cells compared with OCSCC (P = 0.03). The population of mature DCs was lower than the population of immature DCs in all of the tested groups (P < 0.05). There were a greater number of both mature and immature DC populations in the LSCC samples than in the OCSCC, which could contribute to establishing a more effective immune antitumor response for this neoplasm. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effect of female genital schistosomiasis and anti-schistosomal treatment on monocytes, CD4+ T-cells and CCR5 expression in the female genital tract.

PubMed

Kleppa, Elisabeth; Ramsuran, Veron; Zulu, Siphosenkosi; Karlsen, Gunn Hege; Bere, Alfred; Passmore, Jo-Ann S; Ndhlovu, Patricia; Lillebø, Kristine; Holmen, Sigve D; Onsrud, Mathias; Gundersen, Svein Gunnar; Taylor, Myra; Kjetland, Eyrun F; Ndung'u, Thumbi

2014-01-01

Schistosoma haematobium is a waterborne parasite that may cause female genital schistosomiasis (FGS), characterized by genital mucosal lesions. There is clinical and epidemiological evidence for a relationship between FGS and HIV. We investigated the impact of FGS on HIV target cell density and expression of the HIV co-receptor CCR5 in blood and cervical cytobrush samples. Furthermore we evaluated the effect of anti-schistosomal treatment on these cell populations. The study followed a case-control design with post treatment follow-up, nested in an on-going field study on FGS. Blood and cervical cytobrush samples were collected from FGS negative and positive women for flow cytometry analyses. Urine samples were investigated for schistosome ova by microscopy and polymerase chain reaction (PCR). FGS was associated with a higher frequency of CD14+ cells (monocytes) in blood (11.5% in FGS+ vs. 2.2% in FGS-, p = 0.042). Frequencies of CD4+ cells expressing CCR5 were higher in blood samples from FGS+ than from FGS- women (4.7% vs. 1.5%, p = 0.018). The CD14+ cell population decreased significantly in both compartments after anti-schistosomal treatment (p = 0.043). Although the frequency of CD4+ cells did not change after treatment, frequencies of CCR5 expression by CD4+ cells decreased significantly in both compartments (from 3.4% to 0.5% in blood, p = 0.036; and from 42.4% to 5.6% in genital samples, p = 0.025). The results support the hypothesis that FGS may increase the risk of HIV acquisition, not only through damage of the mucosal epithelial barrier, but also by affecting HIV target cell populations, and that anti-schistosomal treatment can modify this.

Aberrant Hypermethylation of SALL3 with HPV Involvement Contributes to the Carcinogenesis of Cervical Cancer.

PubMed

Wei, Xing; Zhang, Shaohua; Cao, Di; Zhao, Minyi; Zhang, Qian; Zhao, Juan; Yang, Ting; Pei, Meili; Wang, Li; Li, Yang; Yang, Xiaofeng

2015-01-01

This study aimed to investigate the methylation status of the promoter region of spalt-like transcription factor 3 (SALL3) and the expression of SALL3 in cervical cancer to explore the function of this gene in cervical cancer carcinogenesis. The methylation status of SALL3 was detected by methylation-specific PCR, and SALL3 gene expression was assessed by real-time quantitative PCR in the cervical cancer cell lines, SiHa, HeLa and C33A, as well as in cervical cancer tissue samples (n = 23), matched pericarcinomatous tissue samples (n = 23) and normal cervix tissue samples (n = 17). MTT was used to measure the cell viability and proliferation capacity of SiHa and HeLa cells. The SALL3 promoter was completely methylated in SiHa cells, unmethylated in C33A cells and partially methylated in HeLa cells. After treatment of SiHa and HeLa cells with 5 μM and 10 μM of 5-Azacytidine (5-Aza), respectively, the methylation level of the SALL3 promoter decreased and observed increase in the degree of unmethylation in a dose-dependent manner. Moreover, the relative expression of SALL3 mRNA increased as the concentration of 5-Aza increased in SiHa (p<0.05) and HeLa (p<0.05) cells. This above-mentioned increase in SALL3 mRNA in SiHa cells was more remarkable than that observed in HeLa cells. Cell proliferation capacity also decreased after administration of 5-Aza to SiHa and HeLa cells (p<0.05). Methylation of the SALL3 promoter was observed in 15 of 23 (65.21%) cervical cancer tissue samples, 15 of 23 (65.21%) matched pericarcinomatous tissue samples and 5 of 17 (29.41%) normal cervical tissue samples (p<0.05). SALL3 mRNA expression was significantly lower in cervical cancer and pericarcinomatous tissues compared with normal cervical tissues (p<0.05). In all cervix tissue samples, HPV infection was positively associated with hypermethylation of the promoter region of SALL3 (p<0.05, r = 0.408), and the expression of SALL3 mRNA in HPV-positive tissues was lower than that in HPV-negative tissues (p<0.05). The aberrant hypermethylation of SALL3 together with HPV involvement inactivated its function as a tumor suppressor and contributed to carcinogenesis in cervical cancer.

Study of wettability and cell viability of H implanted stainless steel

NASA Astrophysics Data System (ADS)

Shafique, Muhammad Ahsan; Ahmad, Riaz; Rehman, Ihtesham Ur

2018-03-01

In the present work, the effect of hydrogen ion implantation on surface wettability and biocompatibility of stainless steel is investigated. Hydrogen ions are implanted in the near-surface of stainless steel to facilitate hydrogen bonding at different doses with constant energy of 500 KeV, which consequently improve the surface wettability. Treated and untreated sample are characterized for surface wettability, incubation of hydroxyapatite and cell viability. Contact angle (CA) study reveals that surface wettability increases with increasing H-ion dose. Raman spectroscopy shows that precipitation of hydroxyapatite over the surface increase with increasing dose of H-ions. Cell viability study using MTT assay describes improved cell viability in treated samples as compared to the untreated sample. It is found that low dose of H-ions is more effective for cell proliferation and the cell count decreases with increasing ion dose. Our study demonstrates that H ion implantation improves the surface wettability and biocompatibility of stainless steel.

Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

PubMed

Thompson, Alison M; Gansen, Alexander; Paguirigan, Amy L; Kreutz, Jason E; Radich, Jerald P; Chiu, Daniel T

2014-12-16

Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

Effects of long-term cryopreservation on peripheral blood progenitor cells.

PubMed

Vosganian, Gregory S; Waalen, Jill; Kim, Kevin; Jhatakia, Sejal; Schram, Ethan; Lee, Tracey; Riddell, Dan; Mason, James R

2012-11-01

The long-term stability of cryopreserved peripheral blood progenitor cells is an important issue for patients experiencing disease relapse. However, there is no consensus on how to evaluate the long-term effects of cryopreservation. We describe the effect of cryopreservation on viability and progenitor colony activity from 87 individual samples processed at the Scripps Green Hospital Stem Cell Processing Center (La Jolla, CA, USA). We randomly selected 87 peripheral blood hematopoietic stem cell (PBHSC) samples from 60 patients and evaluated the effect of cryopreservation on sample viability and red and white cell colony activity after < 24 h and 7, 10 and 15 years of cryopreservation. Viability was assayed via trypan blue dye exclusion and activity was measured following 14 days of culture. An age at collection older than 50 years may result in suboptimal activity and viability following long-term cryopreservation, while gender and disease status had no effect. Cryopreservation did not significantly affect white or red cell activity following 10 years of cryopreservation. However, for samples stored longer than 10 years, viability and activity significantly decreased. We noted a positive association between higher pre-cryopreservation %CD34 count and colony activity. Cryopreservation of peripheral blood progenitor cells for up to 10 years results in no loss of clonogenic capacity, as determined by culture activity, although longer durations of storage may affect activity. Until validated methods are developed, cryopreserved grafts should be evaluated based on pre-freeze CD34(+) cell counts as assayed by flow cytometry, and post-thaw sample evaluation should be reserved for patients identified as poor mobilizers.

Establishment of primary cultures of craniopharyngioma cells★

PubMed Central

Liu, Hao; Liu, Liang; Liu, Zhiyong; Li, Qiang; You, Chao; Xu, Jianguo

2012-01-01

Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research. PMID:25745451

Wavelength-dependent backscattering measurements for quantitative real-time monitoring of apoptosis in living cells

NASA Astrophysics Data System (ADS)

Mulvey, Christine S.; Sherwood, Carly A.; Bigio, Irving J.

2009-11-01

Apoptosis-programmed cell death-is a cellular process exhibiting distinct biochemical and morphological changes. An understanding of the early morphological changes that a cell undergoes during apoptosis can provide the opportunity to monitor apoptosis in tissue, yielding diagnostic and prognostic information. There is avid interest regarding the involvement of apoptosis in cancer. The initial response of a tumor to successful cancer treatment is often massive apoptosis. Current apoptosis detection methods require cell culture disruption. Our aim is to develop a nondisruptive optical method to monitor apoptosis in living cells and tissues. This would allow for real-time evaluation of apoptotic progression of the same cell culture over time without alteration. Elastic scattering spectroscopy (ESS) is used to monitor changes in light-scattering properties of cells in vitro due to apoptotic morphology changes. We develop a simple instrument capable of wavelength-resolved ESS measurements from cell cultures in the backward direction. Using Mie theory, we also develop an algorithm that extracts the size distribution of scatterers in the sample. The instrument and algorithm are validated with microsphere suspensions. For cell studies, Chinese hamster ovary (CHO) cells are cultured to confluence on plates and are rendered apoptotic with staurosporine. Backscattering measurements are performed on pairs of treated and control samples at a sequence of times up to 6-h post-treatment. Initial results indicate that ESS is capable of discriminating between treated and control samples as early as 10- to 15-min post-treatment, much earlier than is sensed by standard assays for apoptosis. Extracted size distributions from treated and control samples show a decrease in Rayleigh and 150-nm scatterers, relative to control samples, with a corresponding increase in 200-nm particles. Work continues to correlate these size distributions with underlying morphology. To our knowledge, this is the first report of the use of backscattering spectral measurements to quantitatively monitor apoptosis in viable cell cultures in vitro.

Cysteine-Rich Intestinal Protein 1 Silencing Inhibits Migration and Invasion in Human Colorectal Cancer.

PubMed

He, Guoyang; Zou, Liyuan; Zhou, Lin; Gao, Peiqiong; Qian, Xinlai; Cui, Jing

2017-01-01

Cysteine-rich intestinal protein 1 (CRIP1), a member of the LIM/double zinc finger protein family, is abnormally expressed in several tumour types. However, few data are available on the role of CRIP1 in cancer. In the present study, we aimed to investigate the expression profile and functions of CRIP1 in colorectal cancer. To examine the protein expression level of CRIP1, immunohistochemistry (IHC) was performed on 56 pairs of colon cancer tissue samples. Western blotting was performed to investigate CRIP1 protein expression in four colon cancer cell lines. The endogenous expression of CRIP1 was suppressed using short interfering RNAs (siRNAs). Cell proliferation assays were used to determine whether CRIP1 silencing affected cell proliferation. Flow cytometry analysis was used to detect cell apoptosis. The effects of silencing CRIP1 on cell migration and invasion was detected using the transwell and wound-healing assays. IHC analysis showed that protein level of CRIP1 was significantly higher in tumour tissue samples than in paired non-tumour tissue samples and that the CRIP1 level was higher in metastatic tissue samples than in non-metastatic tissue samples. In addition, protein levels of CRIP1 were higher in highly metastatic colon cancer cell lines than in colon cancer cell lines with low metastasis. Further, CRIP1 silencing had no effect on cell proliferation or apoptosis in SW620 and HT29 cells. CRIP1 silencing suppressed cell migration and invasion obviously in SW620 and HT29 cells. The present study provides new evidence that abnormal expression of CRIP1 might be related to the degree of metastasis in colorectal cancer and that CRIP1 silencing could effectively inhibit migration and invasion during colorectal cancer development. These findings might aid the development of a biomarker for colon cancer prognosis and metastasis, and thus help to treat this common type of cancer. © 2017 The Author(s). Published by S. Karger AG, Basel.

Evaluation of cell count and classification capabilities in body fluids using a fully automated Sysmex XN equipped with high-sensitive Analysis (hsA) mode and DI-60 hematology analyzer system.

PubMed

Takemura, Hiroyuki; Ai, Tomohiko; Kimura, Konobu; Nagasaka, Kaori; Takahashi, Toshihiro; Tsuchiya, Koji; Yang, Haeun; Konishi, Aya; Uchihashi, Kinya; Horii, Takashi; Tabe, Yoko; Ohsaka, Akimichi

2018-01-01

The XN series automated hematology analyzer has been equipped with a body fluid (BF) mode to count and differentiate leukocytes in BF samples including cerebrospinal fluid (CSF). However, its diagnostic accuracy is not reliable for CSF samples with low cell concentration at the border between normal and pathologic level. To overcome this limitation, a new flow cytometry-based technology, termed "high sensitive analysis (hsA) mode," has been developed. In addition, the XN series analyzer has been equipped with the automated digital cell imaging analyzer DI-60 to classify cell morphology including normal leukocytes differential and abnormal malignant cells detection. Using various BF samples, we evaluated the performance of the XN-hsA mode and DI-60 compared to manual microscopic examination. The reproducibility of the XN-hsA mode showed good results in samples with low cell densities (coefficient of variation; % CV: 7.8% for 6 cells/μL). The linearity of the XN-hsA mode was established up to 938 cells/μL. The cell number obtained using the XN-hsA mode correlated highly with the corresponding microscopic examination. Good correlation was also observed between the DI-60 analyses and manual microscopic classification for all leukocyte types, except monocytes. In conclusion, the combined use of cell counting with the XN-hsA mode and automated morphological analyses using the DI-60 mode is potentially useful for the automated analysis of BF cells.

Optimization of Native and Formaldehyde iPOND Techniques for Use in Suspension Cells.

PubMed

Wiest, Nathaniel E; Tomkinson, Alan E

2017-01-01

The isolation of proteins on nascent DNA (iPOND) technique developed by the Cortez laboratory allows a previously unparalleled ability to examine proteins associated with replicating and newly synthesized DNA in mammalian cells. Both the original, formaldehyde-based iPOND technique and a more recent derivative, accelerated native iPOND (aniPOND), have mostly been performed in adherent cell lines. Here, we describe modifications to both protocols for use with suspension cell lines. These include cell culture, pulse, and chase conditions that optimize sample recovery in both protocols using suspension cells and several key improvements to the published aniPOND technique that reduce sample loss, increase signal to noise, and maximize sample recovery. Additionally, we directly and quantitatively compare the iPOND and aniPOND protocols to test the strengths and limitations of both. Finally, we present a detailed protocol to perform the optimized aniPOND protocol in suspension cell lines. © 2017 Elsevier Inc. All rights reserved.

Method of evaluation of process of red blood cell sedimentation based on photometry of droplet samples.

PubMed

Aristov, Alexander; Nosova, Ekaterina

2017-04-01

The paper focuses on research aimed at creating and testing a new approach to evaluate the processes of aggregation and sedimentation of red blood cells for purpose of its use in clinical laboratory diagnostics. The proposed method is based on photometric analysis of blood sample formed as a sessile drop. The results of clinical approbation of this method are given in the paper. Analysis of the processes occurring in the sample in the form of sessile drop during the process of blood cells sedimentation is described. The results of experimental studies to evaluate the effect of the droplet sample focusing properties on light radiation transmittance are presented. It is shown that this method significantly reduces the sample volume and provides sufficiently high sensitivity to the studied processes.

Possible overestimation of surface disinfection efficiency by assessment methods based on liquid sampling procedures as demonstrated by in situ quantification of spore viability.

PubMed

Grand, I; Bellon-Fontaine, M-N; Herry, J-M; Hilaire, D; Moriconi, F-X; Naïtali, M

2011-09-01

The standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often based on counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldom achieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells are representative of the levels of survivors in whole populations. The present study was thus designed to determine the "damaged/undamaged" status induced by a peracetic acid disinfection for Bacillus atrophaeus spores deposited on glass coupons directly on this substrate and to compare it to the status of spores collected in liquid by a sampling procedure. The method utilized to assess the viability of both surface-associated and liquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyes and quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principal result of the study was that the viability of spores sampled in the liquid was found to be poorer than that of surface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% ± 5% of viable cells were detected among liquid-sampled cells compared to 79% ± 5% or 47% ± 4%, respectively, when the viability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of the survivors collected in the liquid phase, evaluated using the microscopic method and standard plate counts, were well correlated. Evaluations based on the determination of survivors among the liquid-sampled cells can thus overestimate the efficiency of surface disinfection procedures.

Possible Overestimation of Surface Disinfection Efficiency by Assessment Methods Based on Liquid Sampling Procedures as Demonstrated by In Situ Quantification of Spore Viability ▿

PubMed Central

Grand, I.; Bellon-Fontaine, M.-N.; Herry, J.-M.; Hilaire, D.; Moriconi, F.-X.; Naïtali, M.

2011-01-01

The standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often based on counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldom achieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells are representative of the levels of survivors in whole populations. The present study was thus designed to determine the “damaged/undamaged” status induced by a peracetic acid disinfection for Bacillus atrophaeus spores deposited on glass coupons directly on this substrate and to compare it to the status of spores collected in liquid by a sampling procedure. The method utilized to assess the viability of both surface-associated and liquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyes and quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principal result of the study was that the viability of spores sampled in the liquid was found to be poorer than that of surface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% ± 5% of viable cells were detected among liquid-sampled cells compared to 79% ± 5% or 47% ± 4%, respectively, when the viability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of the survivors collected in the liquid phase, evaluated using the microscopic method and standard plate counts, were well correlated. Evaluations based on the determination of survivors among the liquid-sampled cells can thus overestimate the efficiency of surface disinfection procedures. PMID:21742922

The analysis of ALK gene rearrangement by fluorescence in situ hybridization in non-small cell lung cancer patients

PubMed Central

Krawczyk, Paweł Adam; Ramlau, Rodryg Adam; Szumiło, Justyna; Kozielski, Jerzy; Kalinka-Warzocha, Ewa; Bryl, Maciej; Knopik-Dąbrowicz, Alina; Spychalski, Łukasz; Szczęsna, Aleksandra; Rydzik, Ewelina; Milanowski, Janusz

2013-01-01

Introduction ALK gene rearrangement is observed in a small subset (3–7%) of non-small cell lung cancer (NSCLC) patients. The efficacy of crizotinib was shown in lung cancer patients harbouring ALK rearrangement. Nowadays, the analysis of ALK gene rearrangement is added to molecular examination of predictive factors. Aim of the study The frequency of ALK gene rearrangement as well as the type of its irregularity was analysed by fluorescence in situ hybridisation (FISH) in tissue samples from NSCLC patients. Material and methods The ALK gene rearrangement was analysed in 71 samples including 53 histological and 18 cytological samples. The analysis could be performed in 56 cases (78.87%), significantly more frequently in histological than in cytological materials. The encountered problem with ALK rearrangement diagnosis resulted from the scarcity of tumour cells in cytological samples, high background fluorescence noises and fragmentation of cell nuclei. Results The normal ALK copy number without gene rearrangement was observed in 26 (36.62%) patients ALK gene polysomy without gene rearrangement was observed in 25 (35.21%) samples while in 3 (4.23%) samples ALK gene amplification was found. ALK gene rearrangement was observed in 2 (2.82%) samples from males, while in the first case the rearrangement coexisted with ALK amplification. In the second case, signet-ring tumour cells were found during histopathological examination and this patient was successfully treated with crizotinib with partial remission lasting 16 months. Conclusions FISH is a useful technique for ALK gene rearrangement analysis which allows us to specify the type of gene irregularities. ALK gene examination could be performed in histological as well as cytological (cellblocks) samples, but obtaining a reliable result in cytological samples depends on the cellularity of examined materials. PMID:24592134

MixHMM: Inferring Copy Number Variation and Allelic Imbalance Using SNP Arrays and Tumor Samples Mixed with Stromal Cells

PubMed Central

Schulz, Vincent; Chen, Min; Tuck, David

2010-01-01

Background Genotyping platforms such as single nucleotide polymorphism (SNP) arrays are powerful tools to study genomic aberrations in cancer samples. Allele specific information from SNP arrays provides valuable information for interpreting copy number variation (CNV) and allelic imbalance including loss-of-heterozygosity (LOH) beyond that obtained from the total DNA signal available from array comparative genomic hybridization (aCGH) platforms. Several algorithms based on hidden Markov models (HMMs) have been designed to detect copy number changes and copy-neutral LOH making use of the allele information on SNP arrays. However heterogeneity in clinical samples, due to stromal contamination and somatic alterations, complicates analysis and interpretation of these data. Methods We have developed MixHMM, a novel hidden Markov model using hidden states based on chromosomal structural aberrations. MixHMM allows CNV detection for copy numbers up to 7 and allows more complete and accurate description of other forms of allelic imbalance, such as increased copy number LOH or imbalanced amplifications. MixHMM also incorporates a novel sample mixing model that allows detection of tumor CNV events in heterogeneous tumor samples, where cancer cells are mixed with a proportion of stromal cells. Conclusions We validate MixHMM and demonstrate its advantages with simulated samples, clinical tumor samples and a dilution series of mixed samples. We have shown that the CNVs of cancer cells in a tumor sample contaminated with up to 80% of stromal cells can be detected accurately using Illumina BeadChip and MixHMM. Availability The MixHMM is available as a Python package provided with some other useful tools at http://genecube.med.yale.edu:8080/MixHMM. PMID:20532221

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Ming-Hung; School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan; Haung, Chiung-Fang

In this study, neodymium-doped yttrium orthovanadate (Nd:YVO{sub 4}) as a laser source with different scanning speeds was used on biomedical Ti surface. The microstructural and biological properties of laser-modified samples were investigated by means of optical microscope, electron microscope, X-ray diffraction, surface roughness instrument, contact angle and cell cytotoxicity assay. After laser modification, the rough volcano-like recast layer with micro-/nanoporous structure and wave-like recast layer with nanoporous structure were generated on the surfaces of laser-modified samples, respectively. It was also found out that, an α → (α + rutile-TiO{sub 2}) phase transition occurred on the recast layers of laser-modified samples.more » The Ti surface becomes hydrophilic at a high speed laser scanning. Moreover, the cell cytotoxicity assay demonstrated that laser-modified samples did not influence the cell adhesion and proliferation behaviors of osteoblast (MG-63) cell. The laser with 50 mm/s scanning speed induced formation of rough volcano-like recast layer accompanied with micro-/nanoporous structure, which can promote cell adhesion and proliferation of MG-63 cell on Ti surface. The results indicated that the laser treatment was a potential technology to enhance the biocompatibility for titanium. - Highlights: • Laser induced the formation of recast layer with micro-/nanoporous structure on Ti. • An α → (α + rutile-TiO{sub 2}) phase transition was observed within the recast layer. • The Ti surface becomes hydrophilic at a high speed laser scanning. • Laser-modified samples exhibit good biocompatibility to osteoblast (MG-63) cell.« less

Mechanisms of red blood cells agglutination in antibody-treated paper.

PubMed

Jarujamrus, Purim; Tian, Junfei; Li, Xu; Siripinyanond, Atitaya; Shiowatana, Juwadee; Shen, Wei

2012-05-07

Recent reports on using bio-active paper and bio-active thread to determine human blood type have shown a tremendous potential of using these low-cost materials to build bio-sensors for blood diagnosis. In this work we focus on understanding the mechanisms of red blood cell agglutination in the antibody-loaded paper. We semi-quantitatively evaluate the percentage of antibody molecules that are adsorbed on cellulose fibres and can potentially immobilize red blood cells on the fibre surface, and the percentage of the molecules that can desorb from the cellulose fibre surface into the blood sample and cause haemagglutination reaction in the bulk of a blood sample. Our results show that 34 to 42% of antibody molecules in the papers treated with commercial blood grouping antibodies can desorb from the fibre surface. When specific antibody molecules are released into the blood sample via desorption, haemagglutination reaction occurs in the blood sample. The reaction bridges the red cells in the blood sample bulk to the layer of red cells immobilized on the fibre surface by the adsorbed antibody molecules. The desorbed antibody also causes agglutinated lumps of red blood cells to form. These lumps cannot pass through the pores of the filter paper. The immobilization and filtration of agglutinated red cells give reproducible identification of positive haemagglutination reaction. Results from this study provide information for designing new bio-active paper-based devices for human blood typing with improved sensitivity and specificity.

A multicolour flow cytometric assay for c-MYC protein in B-cell lymphoma.

PubMed

Alayed, Khaled; Schweitzer, Karen; Awadallah, Amad; Shetty, Shashirekha; Turakhia, Samir; Meyerson, Howard

2018-05-16

Develop an objective assay to detect c-MYC protein expression using multiparametric flow cytometry (FCM) as an alternative to immunohistochemistry (IHC). 57 patient samples and 11 cell line samples were evaluated. Cell suspensions were obtained and c-MYC staining was performed in combination with CD45 and CD19 and, in some samples, CD10. The percentage of c-MYC+ cells by FCM was correlated with the percentage determined by IHC. The relationship between c-MYC protein expression and the presence of a c-MYC gene rearrangement in aggressive and high-grade lymphomas was also assessed. c-MYC expression by FCM and IHC demonstrated a high degree of correlation in a training set of 33 patient cases, r=0.92, 11 cell line samples, r=0.81 and in a validation set of 24 aggressive and high-grade B-cell lymphomas, r=0.85. c-MYC gene was rearranged by fluorescence in situ hybridisation in 6/9 samples with high c-MYC expression (>40%) by FCM and 6/14 by IHC. We have developed a reliable multicolour FCM assay to detect c-MYC expression suitable for clinical laboratories that should be helpful to accurately quantify c-MYC expression in B-cell lymphomas. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

[Comparison of the Conventional Centrifuged and Filtrated Preparations in Urine Cytology].

PubMed

Sekita, Nobuyuki; Shimosakai, Hirofumi; Nishikawa, Rika; Sato, Hiroaki; Kouno, Hiroyoshi; Fujimura, Masaaki; Mikami, Kazuo

2016-03-01

The urine cytology test is one of the most important tools for the diagnosis of malignant urinary tract tumors. This test is also of great value for predicting malignancy. However, the sensitivity of this test is not high enough to screen for malignant cells. In our laboratory, we were able to attain a high sensitivity of urine cytology tests after changing the preparation method of urine samples. The differences in the cytodiagnosis between the two methods are discussed here. From January 2012 to June 2013, 2,031 urine samples were prepared using the conventional centrifuge method (C method) ; and from September 2013 to March 2015, 2,453 urine samples were prepared using the filtration method (F method) for the cytology test. When the samples included in category 4 or 5, were defined as cytological positive, the sensitivities of this test with samples prepared using the F method were significantly high compared with samples prepared using the C method (72% vs 28%, p＜0.001). The number of cells on the glass slides prepared by the F method was significantly higher than that of the samples prepared by the C method (p＜0.001). After introduction of the F method, the number of f alse negative cases was decreased in the urine cytology test because a larger number of cells was seen and easily detected as atypical or malignant epithelial cells. Therefore, this method has a higher sensitivity than the conventional C method as the sensitivity of urine cytology tests relies partially on the number of cells visualized in the prepared samples.

Detecting cell-in-cell structures in human tumor samples by E-cadherin/CD68/CD45 triple staining

PubMed Central

Wang, Manna; Ning, Xiangkai; He, Meifang; Hu, Yazhuo; Yuan, Long; Li, Shichong; Wang, Qiwei; Liu, Hong; Chen, Zhaolie; Ren, Jun; Sun, Qiang

2015-01-01

Although Cell-in-cell structures (CICs) had been documented in human tumors for decades, it is unclear what types of CICs were formed largely due to low resolution of traditional way such as H&E staining. In this work, we employed immunofluorescent method to stain a panel of human tumor samples simultaneously with antibodies against E-cadherin for Epithelium, CD68 for Macrophage and CD45 for Leukocytes, which we termed as “EML method” based on the cells detected. Detail analysis revealed four types of CICs, with tumor cells or macrophage engulfing tumor cells or leukocytes respectively. Interestingly, tumor cells seem to be dominant over macrophage (93% vs 7%) as the engulfer cells in all CICs detected, whereas the overall amount of internalized tumor cells is comparable to that of internalized CD45+ leukocytes (57% vs 43%). The CICs profiles vary from tumor to tumor, which may indicate different malignant stages and/or inflammatory conditions. Given the potential impacts different types of CICs might have on tumor growth, we therefore recommend EML analysis of tumor samples to clarify the correlation of CICs subtypes with clinical prognosis in future researches. PMID:26109430

Detecting cell-in-cell structures in human tumor samples by E-cadherin/CD68/CD45 triple staining.

PubMed

Huang, Hongyan; Chen, Ang; Wang, Ting; Wang, Manna; Ning, Xiangkai; He, Meifang; Hu, Yazhuo; Yuan, Long; Li, Shichong; Wang, Qiwei; Liu, Hong; Chen, Zhaolie; Ren, Jun; Sun, Qiang

2015-08-21

Although Cell-in-cell structures (CICs) had been documented in human tumors for decades, it is unclear what types of CICs were formed largely due to low resolution of traditional way such as H&E staining. In this work, we employed immunofluorescent method to stain a panel of human tumor samples simultaneously with antibodies against E-cadherin for Epithelium, CD68 for Macrophage and CD45 for Leukocytes, which we termed as "EML method" based on the cells detected. Detail analysis revealed four types of CICs, with tumor cells or macrophage engulfing tumor cells or leukocytes respectively. Interestingly, tumor cells seem to be dominant over macrophage (93% vs 7%) as the engulfer cells in all CICs detected, whereas the overall amount of internalized tumor cells is comparable to that of internalized CD45+ leukocytes (57% vs 43%). The CICs profiles vary from tumor to tumor, which may indicate different malignant stages and/or inflammatory conditions. Given the potential impacts different types of CICs might have on tumor growth, we therefore recommend EML analysis of tumor samples to clarify the correlation of CICs subtypes with clinical prognosis in future researches.

Bioculture System: Expanding ISS Space Bioscience Capabilities for Fundamental Stem Cell Research and Commercial Applications

NASA Astrophysics Data System (ADS)

Blaber, Elizabeth; Dvorochkin, Natalya; Almeida, Eduardo; Fitzpatrick, Garret; Ellingson, Lance; Mitchell, Sarah; Yang, Anthony; Kosnik, Cristine; Rayl, Nicole; Cannon, Tom; Austin, Edward; Sato, Kevin

With the recent call by the 2011 Decadal Report and the 2010 Space Biosciences Roadmap for the International Space Station (ISS) to be used as a National Laboratory for scientific research, there is now a need for new laboratory instruments on ISS to enable such research to occur. The Bioculture System supports the extended culturing of multiple cell types and microbiological specimens. It consists of a docking station that carries ten independent incubation units or ‘Cassettes’. Each Cassette contains a cooling chamber (5(°) C) for temperature sensitive solutions and samples, or long duration fluids and sample storage, as well as an incubation chamber (ambient up to 42(°) C). Each Cassette houses an independent fluidics system comprised of a biochamber, medical-grade fluid tubing, medium warming module, oxygenation module, fluid pump, and sixteen solenoid valves for automated biochamber injections of sampling. The Bioculture System provides the user with the ability to select the incubation temperature, fluid flow rate and automated biochamber sampling or injection events for each separate Cassette. Furthermore, the ISS crew can access the biochamber, media bag, and accessory bags on-orbit using the Microgravity Science Glovebox. The Bioculture System also permits initiation of cultures, subculturing, injection of compounds, and removal of samples for on-orbit processing using ISS facilities. The Bioculture System therefore provides a unique opportunity for the study of stem cells and other cell types in space. The first validation flight of the Bioculture System will be conducted on SpaceX5, consisting of 8 Cassettes and lasting for 30-37 days. During this flight we plan to culture two different mammalian cell types in bioreactors: a mouse osteocytic-like cell line, and human induced pluripotent stem cell (iPS)-derived cardiomyocytes. Specifically, the osteocytic line will enable the study of a type of cell that has been flown on the Bioculture System’s predecessor, the Cell Culture Module, whilst demonstrating the Bioculture Systems bead-based sub-culturing capabilities, automated sampling and fixation, manual sample removal/storage by ISS crew members, and whole bioreactor fixation. These activities will enable, for the first time, the long-duration culture of a proliferative cell line. Furthermore, these activities will facilitate genetic and proteomic analysis of these cells at several time points to determine cell health throughout the culture period. The long-duration culture of iPS-derived cardiomyocytes will afford us the capability to assess the maturation and formation of a cardiac-like tissue in microgravity conditions. Automated sampling of this culture immediately prior to un-berthing from the ISS will enable genetic analysis of the mature cardiomyocyte tissue, whilst still enabling the return of live cultures for analysis of cardiomyocyte morphology, contractility, and viability in response to spaceflight. This validation flight will demonstrate the new functional capabilities of the Bioculture System and the System will enable, for the first time, the study of the response of stem cells and other cell lineages to long-duration spaceflight exposure, whilst enabling normal cell culturing techniques to be automatically conducted on ISS.

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

PubMed

Chan, Leo Li-Ying; Smith, Tim; Kumph, Kendra A; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

2016-10-01

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

Human papillomavirus (HPV) infection in a case-control study of oral squamous cell carcinoma and its increasing trend in northeastern Thailand.

PubMed

Phusingha, Pensiri; Ekalaksananan, Tipaya; Vatanasapt, Patravoot; Loyha, Kulchaya; Promthet, Supannee; Kongyingyoes, Bunkerd; Patarapadungkit, Natcha; Chuerduangphui, Jureeporn; Pientong, Chamsai

2017-06-01

Human papillomavirus (HPV) is an independent risk factor for development of oral squamous cell carcinoma (OSCC). This study aimed to investigate the role of HPV infection and the trend in percentage of HPV-associated OSCC over a 5-year period in northeastern Thailand. In this case-control study, 91 exfoliated oral cell samples and 80 lesion cell samples from OSCC cases and exfoliated oral cells from 100 age/gender-matched controls were collected. HPV infection was investigated by PCR using GP5+/GP6+ primers followed by HPV genotyping using reverse line blot hybridization. Quantitative RT-PCR was used to evaluate HPV oncogene transcription. Temporal trends of HPV infection were evaluated in archived formalin-fixed paraffin-embedded (FFPE) OSCC tissues using in situ hybridization. HPV DNA was found in 17.5% (14/80) of lesion samples from OSCC cases and 29.7% (27/91) of exfoliated oral cell samples from the same cases. These values were significantly higher than in exfoliated oral cell samples from controls (13%, 13/100). HPV-16 was the genotype most frequently found in OSCC cases (92.8%, 13/14 infected cases). Interestingly, HPV oncogene mRNA expression was detected and correlated with OSCC cases (P < 0.005). Of 146 archived FFPE OSCC samples, 82 (56.2%) were positive for high-risk HPV DNA and 64 (43.8%) cases were positive for HPV E6/E7 mRNA expression. There was a trend of increasing percentage of HPV-associated OSCC from 2005 to 2010. This was especially so for females with well-differentiated tumors in specific tongue sub-sites. We suggest that HPV infection plays an important role in oral carcinogenesis in northeastern Thailand. © 2016 Wiley Periodicals, Inc.

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis.

PubMed

Brannelly, Laura A; Roberts, Alexandra A; Skerratt, Lee F; Berger, Lee

2018-05-16

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

LONG-TERM PERFORMANCE OF IN-SITU PERMEABLE REACTIVE BARRIERS FOR REMEDIATION OF CONTAMINATED GROUND WATER

EPA Science Inventory

During this period one sampling trip was performed at the Denver federal site involving EPA and EPA-contractor ManTech) personnel. This occurred in May 1999. During that trip cells 1 and 2 were sampled. Cell 3, although planned to be sampled. was not due to access restrictions. I...

Cutaway line drawing of STS-34 middeck experiment Polymer Morphology (PM)

NASA Technical Reports Server (NTRS)

1989-01-01

Cutaway line drawing shows components of STS-34 middeck experiment Polymer Morphology (PM). Components include the EAC, heat exchanger, sample cell control (SCC), sample cells, source, interferometer, electronics, carousel drive, infrared (IR) beam, and carousel. PM, a 3M-developed organic materials processing experiment, is designed to explore the effects of microgravity on polymeric materials as they are processed in space. The samples of polymeric materials being studied in the PM experiment are thin films (25 microns or less) approximately 25mm in diameter. The samples are mounted between two infrared transparent windows in a specially designed infrared cell that provides the capability of thermally processing the samples to 200 degrees Celsius with a high degree of thermal control. The samples are mounted on a carousel that allows them to be positioned, one at a time, in the infrared beam where spectra may be acquired. The Generic Electronics Module (GEM) provides all carousel and

Tissue-based standoff biosensors for detecting chemical warfare agents

DOEpatents

Greenbaum, Elias; Sanders, Charlene A.

2003-11-18

A tissue-based, deployable, standoff air quality sensor for detecting the presence of at least one chemical or biological warfare agent, includes: a cell containing entrapped photosynthetic tissue, the cell adapted for analyzing photosynthetic activity of the entrapped photosynthetic tissue; means for introducing an air sample into the cell and contacting the air sample with the entrapped photosynthetic tissue; a fluorometer in operable relationship with the cell for measuring photosynthetic activity of the entrapped photosynthetic tissue; and transmitting means for transmitting analytical data generated by the fluorometer relating to the presence of at least one chemical or biological warfare agent in the air sample, the sensor adapted for deployment into a selected area.

[CD4 and CD8 T lymphocytes and NK cells in the stroma of the uterine cervix of women infected with human papillomavirus].

PubMed

Alves, Daniella Borges; Tozetti, Inês Aparecida; Gatto, Flávia Almeida; Cassandri, Fernanda; Ferreira, Alda Maria Teixeira; Carlos Eurico Dos Santos, Fernandes; Falcão, Gustavo Ribeiro; Scapulatempo, Ilzia Doraci Lins; Padovani, Cacilda Tezelli Junqueira; Abdo, Maria Auxiliadora Gomes Sandim

2010-01-01

Immune response might be a key element regarding the progression or regression of human papillomavirus (HPV) infection in the stroma of the uterine cervix. This study aimed to quantify the presence of CD4 and CD8 T lymphocytes and NK cells in the cervical stroma, by means of immunohistochemistry, in high and low grade lesions in patients infected by HPV METHODS: Fifty-six biopsy samples from the uterine cervix were used. Forty-three samples were positive for oncogenic high-risk HPV DNA and had a histopathological diagnosis of high and low-grade cervical intraepithelial neoplasia (CIN) or negative for intraepithelial lesion and malignancy (NILM); while the other 13 samples were negative for HPV DNA with a histopathological diagnosis of NILM RESULTS: Higher quantities of CD4 T lymphocytes were observed in CIN II/III, carcinoma and NILM samples (p = 0.04) and in those in which the viral load was between 10 and 1.000 RLU/PCB. CD8 T lymphocytes were predominant in CIN II/III samples (p = 0.02) and also in samples with viral loads between 100 and 1,000 RLU/PCB. NK cells predominated in samples with low-grade lesions and low viral load This study proved that in the initial stages of the infection, in which no high-grade cell abnormalities have yet occurred, no cells that might trigger the effector phase of the immune response.

In vitro test of external Qigong

PubMed Central

Yount, Garret; Solfvin, Jerry; Moore, Dan; Schlitz, Marilyn; Reading, Melissa; Aldape, Ken; Qian, Yifang

2004-01-01

Background Practitioners of the alternative medical practice 'external Qigong' generally claim the ability to emit or direct "healing energy" to treat patients. We investigated the ability of experienced Qigong practitioners to enhance the healthy growth of cultured human cells in a series of studies, each following a rigorously designed protocol with randomization, blinding and controls for variability. Methods Qigong practitioners directed healing intentionality toward normal brain cell cultures in a basic science laboratory. Qigong treatments were delivered for 20 minutes from a minimum distance of 10 centimeters. Cell proliferation was measured by a standard colony-forming efficiency (CFE) assay and a CFE ratio (CFE for treated samples/CFE for sham samples) was the dependent measure for each experiment. Results During a pilot study (8 experiments), a trend of increased cell proliferation in Qigong-treated samples (CFE Qigong/sham ratios > 1.0) was observed (P = 0.162). In a formal study (28 experiments), a similar trend was observed, with Qigong-treated samples showing on average more colony formation than sham samples (P = 0.036). In a replication study (60 experiments), no significant difference between Qigong-treated samples and sham samples was observed (P = 0.465). Conclusion We observed an apparent increase in the proliferation of cultured cells following external Qigong treatment by practitioners under strictly controlled conditions, but we did not observe this effect in a replication study. These results suggest the need for more controlled and thorough investigation of external Qigong before scientific validation is claimed. PMID:15102336

Adipose tissue-derived stem cell response to the differently processed 316L stainless steel substrates.

PubMed

Faghihi, Shahab; Zia, Sonia; Taha, Masoumeh Fakhr

2012-12-01

Stainless steel (SS) is one of the most applicable materials in fabrication of cardiac implants. The aim of this study is to investigate the effect of atomic structure of polycrystalline stainless steel on the response of adipose tissue-derived stem cells (ADSCs). Samples are prepared from differently processed extruded rod and rolled sheet of 316L SS having different crystallographic structure. X-ray diffraction analysis indicated (200) and (111) orientations with distinct volume fractions in the specimens. Morphology and ADSCs behavior including adhesion, proliferation and differentiation are assessed. The expression of cardiac specific protein (cardiac troponin I) and genes of differentiating cardiomyocytes is analyzed by immunofluorescence and RT-PCR. The number of attached and grown cells on the rod sample is higher than the sheet sample also the scanning electron microscopy (SEM) analysis of ADSCs grown on the samples demonstrates higher cell density and spreading pattern on the surface of rod sample. In differentiated ADSCs on the rod sample the expression of all genes except ANF are detectable, while on the sheet sample only the MEF2C and β-MHC are expressed. This study shows that the cellular response is influenced by the crystal structure of the substrate therefore; the skill to alter the structure of substrate may lend itself to engineer a biomaterial which could be suitable for differentiation of stem cells into a definite lineage. Copyright © 2012 Elsevier Ltd. All rights reserved.

Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing.

PubMed

Watanabe, Manabu; Kusano, Junko; Ohtaki, Shinsaku; Ishikura, Takashi; Katayama, Jin; Koguchi, Akira; Paumen, Michael; Hayashi, Yoshiharu

2014-09-01

Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line) were used as a model. Single-cell capture was performed using laser capture microdissection (LCM) with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈ 10(6) cells) were subjected to whole genome amplification (WGA). For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel) was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 10(31-35). For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100 × were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100 × were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.

Diffusing-wave spectroscopy in a standard dynamic light scattering setup

NASA Astrophysics Data System (ADS)

Fahimi, Zahra; Aangenendt, Frank J.; Voudouris, Panayiotis; Mattsson, Johan; Wyss, Hans M.

2017-12-01

Diffusing-wave spectroscopy (DWS) extends dynamic light scattering measurements to samples with strong multiple scattering. DWS treats the transport of photons through turbid samples as a diffusion process, thereby making it possible to extract the dynamics of scatterers from measured correlation functions. The analysis of DWS data requires knowledge of the path length distribution of photons traveling through the sample. While for flat sample cells this path length distribution can be readily calculated and expressed in analytical form; no such expression is available for cylindrical sample cells. DWS measurements have therefore typically relied on dedicated setups that use flat sample cells. Here we show how DWS measurements, in particular DWS-based microrheology measurements, can be performed in standard dynamic light scattering setups that use cylindrical sample cells. To do so we perform simple random-walk simulations that yield numerical predictions of the path length distribution as a function of both the transport mean free path and the detection angle. This information is used in experiments to extract the mean-square displacement of tracer particles in the material, as well as the corresponding frequency-dependent viscoelastic response. An important advantage of our approach is that by performing measurements at different detection angles, the average path length through the sample can be varied. For measurements performed on a single sample cell, this gives access to a wider range of length and time scales than obtained in a conventional DWS setup. Such angle-dependent measurements also offer an important consistency check, as for all detection angles the DWS analysis should yield the same tracer dynamics, even though the respective path length distributions are very different. We validate our approach by performing measurements both on aqueous suspensions of tracer particles and on solidlike gelatin samples, for which we find our DWS-based microrheology data to be in good agreement with rheological measurements performed on the same samples.

Spatial-Resolution Cell Type Proteome Profiling of Cancer Tissue by Fully Integrated Proteomics Technology.

PubMed

Xu, Ruilian; Tang, Jun; Deng, Quantong; He, Wan; Sun, Xiujie; Xia, Ligang; Cheng, Zhiqiang; He, Lisheng; You, Shuyuan; Hu, Jintao; Fu, Yuxiang; Zhu, Jian; Chen, Yixin; Gao, Weina; He, An; Guo, Zhengyu; Lin, Lin; Li, Hua; Hu, Chaofeng; Tian, Ruijun

2018-05-01

Increasing attention has been focused on cell type proteome profiling for understanding the heterogeneous multicellular microenvironment in tissue samples. However, current cell type proteome profiling methods need large amounts of starting materials which preclude their application to clinical tumor specimens with limited access. Here, by seamlessly combining laser capture microdissection and integrated proteomics sample preparation technology SISPROT, specific cell types in tumor samples could be precisely dissected with single cell resolution and processed for high-sensitivity proteome profiling. Sample loss and contamination due to the multiple transfer steps are significantly reduced by the full integration and noncontact design. H&E staining dyes which are necessary for cell type investigation could be selectively removed by the unique two-stage design of the spintip device. This easy-to-use proteome profiling technology achieved high sensitivity with the identification of more than 500 proteins from only 0.1 mm 2 and 10 μm thickness colon cancer tissue section. The first cell type proteome profiling of four cell types from one colon tumor and surrounding normal tissue, including cancer cells, enterocytes, lymphocytes, and smooth muscle cells, was obtained. 5271, 4691, 4876, and 2140 protein groups were identified, respectively, from tissue section of only 5 mm 2 and 10 μm thickness. Furthermore, spatially resolved proteome distribution profiles of enterocytes, lymphocytes, and smooth muscle cells on the same tissue slices and across four consecutive sections with micrometer distance were successfully achieved. This fully integrated proteomics technology, termed LCM-SISPROT, is therefore promising for spatial-resolution cell type proteome profiling of tumor microenvironment with a minute amount of clinical starting materials.

EZH2 and CD79B mutational status over time in B-cell non-Hodgkin lymphomas detected by high-throughput sequencing using minimal samples

PubMed Central

Saieg, Mauro Ajaj; Geddie, William R; Boerner, Scott L; Bailey, Denis; Crump, Michael; da Cunha Santos, Gilda

2013-01-01

BACKGROUND: Numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHLs) have been revealed by novel high-throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex-associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B-cell NHLs, through use of a customized multiplex mutation assay. METHODS: DNA that was extracted from cytological material stored on FTA cards as well as from additional specimens, including archived frozen and formalin-fixed histological specimens, archived stained smears, and cytospin preparations, were submitted to a multiplex mutation assay specifically designed for the detection of point mutations involving EZH2 and CD79B, using MassARRAY spectrometry followed by Sanger sequencing. RESULTS: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y646) and CD79B (Y196) were detected in 13.2% and 8% of the samples, respectively, almost exclusively in follicular lymphomas and diffuse large B-cell lymphomas. In one-third of the positive cases, a wild type was detected in a different sample from the same patient during follow-up. CONCLUSIONS: Testing multiple minimal tissue samples using a high-throughput multiplex platform exponentially increases tissue availability for molecular analysis and might facilitate future studies of tumor progression and the related molecular events. Mutational status of EZH2 and CD79B may vary in B-cell NHL samples over time and support the concept that individualized therapy should be based on molecular findings at the time of treatment, rather than on results obtained from previous specimens. Cancer (Cancer Cytopathol) 2013;121:377–386. © 2013 American Cancer Society. PMID:23361872

Exclusive Liquid Repellency: An Open Multi-Liquid-Phase Technology for Rare Cell Culture and Single-Cell Processing.

PubMed

Li, Chao; Yu, Jiaquan; Schehr, Jennifer; Berry, Scott M; Leal, Ticiana A; Lang, Joshua M; Beebe, David J

2018-05-23

The concept of high liquid repellency in multi-liquid-phase systems (e.g., aqueous droplets in an oil background) has been applied to areas of biomedical research to realize intrinsic advantages not available in single-liquid-phase systems. Such advantages have included minimizing analyte loss, facile manipulation of single-cell samples, elimination of biofouling, and ease of use regarding loading and retrieving of the sample. In this paper, we present generalized design rules for predicting the wettability of solid-liquid-liquid systems (especially for discrimination between exclusive liquid repellency (ELR) and finite liquid repellency) to extend the applications of ELR. We then apply ELR to two model systems with open microfluidic design in cell biology: (1) in situ underoil culture and combinatorial coculture of mammalian cells in order to demonstrate directed single-cell multiencapsulation with minimal waste of samples as compared to stochastic cell seeding and (2) isolation of a pure population of circulating tumor cells, which is required for certain downstream analyses including sequencing and gene expression profiling.

Effect of surface pre-treatments on biocompatibility of magnesium.

PubMed

Lorenz, Carla; Brunner, Johannes G; Kollmannsberger, Philip; Jaafar, Leila; Fabry, Ben; Virtanen, Sannakaisa

2009-09-01

This study reports the influence of Mg surface passivation on the survival rate of human HeLa cells and mouse fibroblasts in cell culture experiments. Polished samples of commercially pure Mg show high reactivity in the cell culture medium, leading to a pH shift in the alkaline direction, and therefore cell adhesion and survival is strongly impaired. Passivation of the Mg surface in 1M NaOH can strongly enhance cell survival. The best initial cell adhesion is observed for Mg samples incubated in simulated body fluid (M-SBF), which leads to the formation of a biomimetic, amorphous Ca/Mg-phosphate layer with high surface roughness. This surface layer, however, passivates and seals the Mg surface only partially. Subsequent Mg dissolution leads to a significantly stronger pH increase compared to NaOH-passivated samples, which prevents long-term cell survival. These results demonstrate that surface passivation with NaOH and M-SBF together with the associated changes of surface reactivity, chemistry and roughness provide a viable strategy to facilitate cell survival on otherwise non-biocompatible Mg surfaces.

Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

PubMed Central

Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

2013-01-01

A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube. PMID:23874571

Somatic cell counts in bulk milk and their importance for milk processing

NASA Astrophysics Data System (ADS)

Savić, N. R.; Mikulec, D. P.; Radovanović, R. S.

2017-09-01

Bulk tank milk somatic cell counts are the indicator of the mammary gland health in the dairy herds and may be regarded as an indirect measure of milk quality. Elevated somatic cell counts are correlated with changes in milk composition The aim of this study was to assess the somatic cell counts that significantly affect the quality of milk and dairy products. We examined the somatic cell counts in bulk tank milk samples from 38 farms during the period of 6 months, from December to the May of the next year. The flow cytometry, Fossomatic was used for determination of somatic cell counts. In the same samples content of total proteins and lactose was determined by Milcoscan. Our results showed that average values for bulk tank milk samples were 273,605/ml from morning milking and 292,895/ml from evening milking. The average values for total proteins content from morning and evening milking are 3,31 and 3,34%, respectively. The average values for lactose content from morning and evening milking are 4,56 and 4,63%, respectively. The highest somatic cell count (516,000/ml) was detected in bulk tank milk sample from evening milk in the Winter and the lowest content of lactose was 4,46%. Our results showed that obtained values for bulk tank milk somatic cell counts did not significantly affected the content of total proteins and lactose.

Continuous monitoring of L-glutamate released from cultured nerve cells by an online sensor coupled with micro-capillary sampling.

PubMed

Niwa, O; Horiuchi, T; Torimitsu, K

1997-01-01

A small volume L-glutamate online sensor was developed in order to monitor changes in the local concentration of L-glutamate released from cultured nerve cells. Syringe pump in the suction mode is used to sample extracellular fluid continuously from a glass micro-capillary and the concentration of L-glutamate can be determined by using a glassy carbon (GC) electrode modified with an Os-polyvinylpyridine mediator bottom film containing horseradish peroxidase and a bovine serum albumin top layer containing L-glutamate oxidase. The overall efficiency of L-glutamate detection with a sensor is 71% under optimum conditions due to an efficient enzymatic reaction at the modified electrode in the thin layer radial flow cell. As a result, we achieved a detection limit of 7-15 nM and a linear range of 50 nM to 10 microM. In an in vitro experiment, the extracellular fluid near a particular nerve cell can be sampled with this micro-pipet and continuously introduced into the modified GC electrode in the radial flow cell via suction provided by a syringe pump. The nerve cells are stimulated by the KCl in a glass capillary and the L-glutamate concentration change can be monitored by changing the distance between the sampling pipet and the nerve cells.

Impact of lithium alone or in combination with haloperidol on oxidative stress parameters and cell viability in SH-SY5Y cell culture.

PubMed

Gawlik-Kotelnicka, Oliwia; Mielicki, Wojciech; Rabe-Jabłońska, Jolanta; Lazarek, Jerry; Strzelecki, Dominik

2016-02-01

It has been reported that lithium may inhibit lipid peroxidation and protein oxidation. Lithium salts also appear to stimulate cell proliferation, increase neurogenesis, and delay cell death. Oxidative stress and neurodegeneration may play an important role in the pathophysiology of bipolar disorder and the disease course thereof. The aim of this research is to estimate the influence of lithium (alone and in combination with haloperidol) on the parameters of oxidative stress and viability of SH-SY5Y cell lines in neutral and pro-oxidative conditions. The evaluated oxidative stress parameter was lipid peroxidation. The viability of the cell lines was measured utilising the MTT test. In neutral conditions, higher levels of thiobarbituric acid reactive substances were observed in those samples which contained both haloperidol and lithium than in other samples. However, these differences were not statistically significant. Cell viability was significantly higher in therapeutic lithium samples than in the controls; samples of haloperidol alone as well as those of haloperidol with lithium did not differ from controls. The results of our study may indicate that lithium possess neuroprotective properties that may be partly due to antioxidative effects. The combination of lithium and haloperidol may generate increased oxidative stress.

Intragraft Molecular Pathways Associated with Tolerance Induction in Renal Transplantation.

PubMed

Gallon, Lorenzo; Mathew, James M; Bontha, Sai Vineela; Dumur, Catherine I; Dalal, Pranav; Nadimpalli, Lakshmi; Maluf, Daniel G; Shetty, Aneesha A; Ildstad, Suzanne T; Leventhal, Joseph R; Mas, Valeria R

2018-02-01

The modern immunosuppression regimen has greatly improved short-term allograft outcomes but not long-term allograft survival. Complications associated with immunosuppression, specifically nephrotoxicity and infection risk, significantly affect graft and patient survival. Inducing and understanding pathways underlying clinical tolerance after transplantation are, therefore, necessary. We previously showed full donor chimerism and immunosuppression withdrawal in highly mismatched allograft recipients using a bioengineered stem cell product (FCRx). Here, we evaluated the gene expression and microRNA expression profiles in renal biopsy samples from tolerance-induced FCRx recipients, paired donor organs before implant, and subjects under standard immunosuppression (SIS) without rejection and with acute rejection. Unlike allograft samples showing acute rejection, samples from FCRx recipients did not show upregulation of T cell- and B cell-mediated rejection pathways. Gene expression pathways differed slightly between FCRx samples and the paired preimplantation donor organ samples, but most of the functional gene networks overlapped. Notably, compared with SIS samples, FCRx samples showed upregulation of genes involved in pathways, like B cell receptor signaling. Additionally, prediction analysis showed inhibition of proinflammatory regulators and activation of anti-inflammatory pathways in FCRx samples. Furthermore, integrative analyses (microRNA and gene expression profiling from the same biopsy sample) identified the induction of regulators with demonstrated roles in the downregulation of inflammatory pathways and maintenance of tissue homeostasis in tolerance-induced FCRx samples compared with SIS samples. This pilot study highlights the utility of molecular intragraft evaluation of pathways related to FCRx-induced tolerance and the use of integrative analyses for identifying upstream regulators of the affected downstream molecular pathways. Copyright © 2018 by the American Society of Nephrology.

Preparation of pure microbiological samples for pyrolysis of gas-liquid chromatography studies.

PubMed Central

Oxborrow, G S; Fields, N D; Puleo, J R

1976-01-01

Bacterial samples were prepared for pyrolysis gas-liquid chromatography using cells grown on membrane filters. Pyrochromatograms were reproducible when cells harvested from the filters were pyrolyzed without being washed. Images PMID:970947

Foam generation and sample composition optimization for the FOAM-C experiment of the ISS

NASA Astrophysics Data System (ADS)

Carpy, R.; Picker, G.; Amann, B.; Ranebo, H.; Vincent-Bonnieu, S.; Minster, O.; Winter, J.; Dettmann, J.; Castiglione, L.; Höhler, R.; Langevin, D.

2011-12-01

End of 2009 and early 2010 a sealed cell, for foam generation and observation, has been designed and manufactured at Astrium Friedrichshafen facilities. With the use of this cell, different sample compositions of "wet foams" have been optimized for mixtures of chemicals such as water, dodecanol, pluronic, aethoxisclerol, glycerol, CTAB, SDS, as well as glass beads. This development is performed in the frame of the breadboarding development activities of the Experiment Container FOAM-C for operation in the ISS Fluid Science Laboratory (ISS). The sample cell supports multiple observation methods such as: Diffusing-Wave and Diffuse Transmission Spectrometry, Time Resolved Correlation Spectroscopy [1] and microscope observation, all of these methods are applied in the cell with a relatively small experiment volume <3cm3. These units, will be on orbit replaceable sets, that will allow multiple sample compositions processing (in the range of >40).

Optimization of a Viability PCR Method for the Detection of Listeria monocytogenes in Food Samples.

PubMed

Agustí, Gemma; Fittipaldi, Mariana; Codony, Francesc

2018-06-01

Rapid detection of Listeria and other microbial pathogens in food is an essential part of quality control and it is critical for ensuring the safety of consumers. Culture-based methods for detecting foodborne pathogens are time-consuming, laborious and cannot detect viable but non-culturable microorganism, whereas viability PCR methodology provides quick results; it is able to detect viable but non-culturable cells, and allows for easier handling of large amount of samples. Although the most critical point to use viability PCR technique is achieving the complete exclusion of dead cell amplification signals, many improvements are being introduced to overcome this. In the present work, the yield of dead cell DNA neutralization was enhanced by incorporating two new sample treatment strategies: tube change combined with a double light treatment. This procedure was successfully tested using artificially contaminated food samples, showing improved neutralization of dead cell DNA.

Neon and Helium in the Surface of Stardust Cell C2028

NASA Technical Reports Server (NTRS)

Palma, R. L.; Pepin, R. O.; Schlutter, D. J.; Frank, D. R.; Bastien, R.; Rodriguez, M.

2015-01-01

Previous studies of light noble gases in Stardust aerogel samples detected a variety of isotopically non-terrestrial He and Ne compositions. However, with one exception, in none of these samples was there visible evidence for the presence of particles that could have hosted the gases. The exception is materials keystoned from track 41, cell C2044, which contained observable fragments of the impacting Wild 2 comet coma grain. Here we report noble gas data from a second aerogel sample in which grains are observed, cut from the surface of a cell (C2028) riddled with tiny tracks and particles that are thought to be secondary in origin, ejected toward the cell when a parent grain collided with the spacecraft structure and fragmented. Interestingly, measured 20Ne/22Ne ratios in the track 41 and C2028 samples are similar, and within error of the meteoritic "Q-phase" Ne composition.

Identification of immunophenotypic subtypes with different prognoses in extranodal natural killer/T-cell lymphoma, nasal type.

PubMed

Yu, Jian-Bo; Zuo, Zhuo; Zhang, Wen-Yan; Yang, Qun-Pei; Zhang, Ying-Chun; Tang, Yuan; Zhao, Sha; Mo, Xian-Ming; Liu, Wei-Ping

2014-11-01

To analyze the differentiation characteristics of extranodal natural killer/T-cell lymphoma, nasal type, one nude mouse model, cell lines SNK6 and SNT8, and 16 fresh human samples were analyzed by flow cytometry immunophenotyping and immunohistochemistry staining; and 115 archived cases were used for phenotypic detection and prognostic analysis. We found that CD25 was expressed by most tumor cells in all samples, and CD56(+)CD25(+) cells were the predominant population in the mouse model, the 2 cell lines, and 10 of the 16 fresh tumor samples; in the other 6 fresh tumor samples, the predominant cell population was of the CD16(+)CD25(+) phenotype, and only a minor population showed the CD56(+)CD25(+) phenotype. The phenotype detected by immunohistochemistry staining generally was consistent with the phenotype found by flow cytometry immunophenotyping. According to the expression of CD56 and CD16, 115 cases could be classified into 3 phenotypic subtypes: CD56(-)CD16(-), CD56(+)CD16(-), and CD56(dim/-)CD16(+). Patients with tumors of the CD56(dim/-)CD16(+) phenotype had a poorer prognosis than patients with tumors of the other phenotypes. Differentiation of extranodal natural killer/T-cell lymphoma, nasal type apparently resembles the normal natural killer cell developmental pattern, and these tumors can be classified into 3 phenotypic subtypes of different aggressiveness. Expression of CD56(dim/-)CD16(+) implies a poorer prognosis. Copyright © 2014 Elsevier Inc. All rights reserved.

Determining baselines and variability of elements in plants and soils near the Kenai National Wildlife Refuge, Alaska

USGS Publications Warehouse

Crock, J.G.; Severson, R.C.; Gough, L.P.

1992-01-01

Recent investigations on the Kenai Peninsula had two major objectives: (1) to establish elemental baseline concentrations ranges for native vegetation and soils; and, (2) to determine the sampling density required for preparing stable regional geochemical maps for various elements in native plants and soils. These objectives were accomplished using an unbalanced, nested analysis-of-variance (ANOVA) barbell sampling design. Hylocomium splendens (Hedw.) BSG (feather moss, whole plant), Picea glauca (Moench) Voss (white spruce, twigs and needles), and soil horizons (02 and C) were collected and analyzed for major and trace total element concentrations. Using geometric means and geometric deviations, expected baseline ranges for elements were calculated. Results of the ANOVA show that intensive soil or plant sampling is needed to reliably map the geochemistry of the area, due to large local variability. For example, producing reliable element maps of feather moss using a 50 km cell (at 95% probability) would require sampling densities of from 4 samples per cell for Al, Co, Fe, La, Li, and V, to more than 15 samples per cell for Cu, Pb, Se, and Zn.Recent investigations on the Kenai Peninsula had two major objectives: (1) to establish elemental baseline concentrations ranges for native vegetation and soils; and, (2) to determine the sampling density required for preparing stable regional geochemical maps for various elements in native plants and soils. These objectives were accomplished using an unbalanced, nested analysis-of-variance (ANOVA) barbell sampling design. Hylocomium splendens (Hedw.) BSG (feather moss, whole plant), Picea glauca (Moench) Voss (white spruce, twigs and needles), and soil horizons (02 and C) were collected and analyzed for major and trace total element concentrations. Using geometric means and geometric deviations, expected baseline ranges for elements were calculated. Results of the ANOVA show that intensive soil or plant sampling is needed to reliably map the geochemistry of the area, due to large local variability. For example, producing reliable element maps of feather moss using a 50 km cell (at 95% probability) would require sampling densities of from 4 samples per cell Al, Co, Fe, La, Li, and V, to more than 15 samples per cell for Cu, Pb, Se, and Zn.

Identification of PM{sub 10} characteristics involved in cellular responses in human bronchial epithelial cells (Beas-2B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Den Heuvel, Rosette, E-mail: rosette.vandenheuvel@vito.be; Den Hond, Elly, E-mail: elly.denhond@wiv-isp.be; Govarts, Eva, E-mail: eva.govarts@vito.be

Notwithstanding evidence is present that physicochemical characteristics of ambient particles attribute to adverse health effects, there is still some lack of understanding in this complex relationship. At this moment it is not clear which properties (such as particle size, chemical composition) or sources of the particles are most relevant for health effects. This study investigates the in vitro toxicity of PM{sub 10} in relation to PM chemical composition, black carbon (BC), endotoxin content and oxidative potential (OP). In 2013–2014 PM{sub 10} was sampled (24 h sampling, 108 sampling days) in ambient air at three sites in Flanders (Belgium) with differentmore » pollution characteristics: an urban traffic site (Borgerhout), an industrial area (Zelzate) and a rural background location (Houtem). To characterize the toxic potential of PM{sub 10}, airway epithelial cells (Beas-2B cells) have been exposed to particles in vitro. Different endpoints were studied including cell damage and death (cell viability) using the Neutral red Uptake assay, the production of pro-inflammatory molecules by interleukin 8 (IL-8) induction and DNA-damaging activity using the FPG-modified Comet assay. The endotoxin levels in the collected samples were analysed and the capacity of PM{sub 10} particles to produce reactive oxygen species (OP) was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Chemical characteristics of PM{sub 10} (BC, As, Cd, Cr, Cu, Mn, Ni, Pb, Zn) and meteorological conditions were recorded on the sampling days. PM{sub 10} particles exhibited dose-dependent cytotoxicity in Beas-2B cells and were found to significantly induce the release of IL-8 in samples from the three locations. Oxidatively damaged DNA was observed in exposed Beas-2B cells. Endotoxin levels above the detection limit were detected in half of the samples. OP was measurable in all samples. Associations between PM{sub 10} characteristics and biological effects of PM{sub 10} were assessed by single and multiple regression analyses. The reduction in cell viability was significantly correlated with BC, Cd and Pb. The induction of IL-8 in Beas-2B cells was significantly associated with Cu, Ni and Zn and endotoxin. Endotoxin levels explained 33% of the variance in IL-8 induction. A significant interaction between ambient temperature and endotoxin on the pro-inflammatory activity was seen. No association was found between OP and the cellular responses. This study supports the hypothesis that, on an equal mass basis, PM{sub 10} induced biological effects differ due to differences in PM{sub 10} characteristics. Metals (Cd, Cu, Ni and Zn), BC, and endotoxin were among the main determinants for the observed biological responses. - Highlights: • On an equal mass basis, PM{sub 10} sampled at an urban, rural and industrial site induced different cellular effects in Beas-2B. • Endotoxin levels and oxidative potential (OP) were analysed in the PM{sub 10} samples. • Black carbon, cadmium and lead were correlated with decreased cell viability. • Endotoxin levels explained the majority of the variance in il-8 induction. • Oxidatively damaged DNA was observed in all the samples.« less

Multivariate classification of the infrared spectra of cell and tissue samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haaland, D.M.; Jones, H.D.; Thomas, E.V.

1997-03-01

Infrared microspectroscopy of biopsied canine lymph cells and tissue was performed to investigate the possibility of using IR spectra coupled with multivariate classification methods to classify the samples as normal, hyperplastic, or neoplastic (malignant). IR spectra were obtained in transmission mode through BaF{sub 2} windows and in reflection mode from samples prepared on gold-coated microscope slides. Cytology and histopathology samples were prepared by a variety of methods to identify the optimal methods of sample preparation. Cytospinning procedures that yielded a monolayer of cells on the BaF{sub 2} windows produced a limited set of IR transmission spectra. These transmission spectra weremore » converted to absorbance and formed the basis for a classification rule that yielded 100{percent} correct classification in a cross-validated context. Classifications of normal, hyperplastic, and neoplastic cell sample spectra were achieved by using both partial least-squares (PLS) and principal component regression (PCR) classification methods. Linear discriminant analysis applied to principal components obtained from the spectral data yielded a small number of misclassifications. PLS weight loading vectors yield valuable qualitative insight into the molecular changes that are responsible for the success of the infrared classification. These successful classification results show promise for assisting pathologists in the diagnosis of cell types and offer future potential for {ital in vivo} IR detection of some types of cancer. {copyright} {ital 1997} {ital Society for Applied Spectroscopy}« less

Laser capture microdissection: should an ultraviolet or infrared laser be used?

PubMed

Vandewoestyne, Mado; Goossens, Karen; Burvenich, Christian; Van Soom, Ann; Peelman, Luc; Deforce, Dieter

2013-08-15

Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation, however, are crucial for the success of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user-friendliness to sample quality. The comparison was performed on two types of samples: peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. Not only does the UV system allow faster and more precise sample collection, but also the obtained samples-even single cell samples-can be used for DNA extraction and downstream polymerase chain reaction (PCR) applications. RNA-based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10 cells for reverse transcription quantitative PCR (RT-qPCR) analysis, the low RNA quality should be taken into account when designing the RT-qPCR assays. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens.

PubMed

Kim, Dong-Young; Sato, Ayuko; Fukuyama, Satoshi; Sagara, Hiroshi; Nagatake, Takahiro; Kong, Il Gyu; Goda, Kaoru; Nochi, Tomonori; Kunisawa, Jun; Sato, Shintaro; Yokota, Yoshifumi; Lee, Chul Hee; Kiyono, Hiroshi

2011-04-01

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.

Diagnostic value of JC/BK virus antibody immunohistochemistry staining in urine samples from posttransplant immunosuppressed patients in relation to polyomavirus reactivation.

PubMed

Yuste, Rosario Sanchez; Frías, Carolina; López, Ana; Vallejo, Carlos; Martín, Paloma; Bellas, Carmen

2008-01-01

To compare the diagnostic value of cytology and immunohistochemistry staining (IHS) of urine samples for polyomavirus reactivation diagnosis. Sixty-eight urine samples collected from 18 immunosuppressed patients were analyzed by Papanicolaou and IHS with a JC/BK virus-specific monoclonal antibody. Overall, polyomavirus BK (BKV) was positive in 11 of 18 patients (61.1%) (3 of whom developed hemorrhagic cystitis) and in 23 of 68 urine samples (28%). Of 23 samples, 4 (17%) were positive by 1 of the 2 techniques, only. Of 23 samples, 19 (83%) were positive by both methods. In matching urine samples from the same patient, the number of BKV-infected positive cells detected by IHS in urine slides was higher than those detected by Papanicolaou staining (71.3%). The main advantage of LHS is that it allowed confirmation of BKV infection diagnosis in urine samples. IHS detected more BKV-infected cells in samples with few positive urothelial cells, which would have gone undetected if only Papanicolaou staining had been used as the BKV screening method. Urine samples testing for BKV by both techniques will improve diagnosis in asymptomatic patients, allowing early therapeutic intervention and a better clinical outcome.

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

PubMed Central

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-01-01

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice. PMID:28425472

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system.

PubMed

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-04-20

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice.

Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

PubMed

Lebaron, P; Servais, P; Agogué, H; Courties, C; Joux, F

2001-04-01

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?

PubMed Central

Lebaron, Philippe; Servais, Pierre; Agogué, Helene; Courties, Claude; Joux, Fabien

2001-01-01

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems. PMID:11282632

Evidence for apoptosis in human atherogenesis and in a rat vascular injury model.

PubMed Central

Han, D. K.; Haudenschild, C. C.; Hong, M. K.; Tinkle, B. T.; Leon, M. B.; Liau, G.

1995-01-01

Apoptosis is a physiological cell death process important for normal development and involved in many pathological conditions. In atherosclerosis, pathological accumulation of cells in the intima has been attributed to the migration and proliferation of smooth muscle cells, macrophages, and lymphocytes. In this report, we explored the possibility that apoptosis may also contribute to the pathogenesis of this disease. We examined 35 human atherosclerotic lesion samples and identified a substantial number of cells undergoing apoptosis in 25 of the samples. Furthermore, in a rat vascular injury model, apoptotic cells were specifically identified in the neointima. The presence of apoptotic cells was demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, nuclear staining with propidium iodide, and electron microscopy. Immunostaining with cell-type-specific markers and subsequent terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling analysis on the same sample revealed that the majority of the apoptotic cells were modulated smooth muscle cells as well as macrophages. These results indicate that apoptosis occurs in cells of the injured blood vessel as well as the advanced atherosclerotic lesion and that physiological cell death may have an important role in determining the course of atherogenesis. Images Figure 1 Figure 2 Figure 4 Figure 5 PMID:7639326

The effect of space flight on monoclonal antibody synthesis in a hybridoma mouse cell line

NASA Technical Reports Server (NTRS)

Smiley, S. A.; Gillock, E. T.; Black, M. C.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1997-01-01

The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [35S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.

Split rheometer Couette attachment to enable sample extraction

NASA Astrophysics Data System (ADS)

Guthrie, Sarah E.; Idziak, Stefan H. J.

2005-02-01

We report on the development of a Couette attachment insert for a rheometer, which is designed to split in half, enabling intact sample extraction of cocoa butter crystallized from the melt under known dynamic stress conditions. This cell is capable of producing a sample 1mm thick. At shear rates of 90-720s-1 and final temperatures of 18-20°C it was shown that the sample will completely separate from the cell surface intact.

Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects.

PubMed

Nishiyama, Kazuhiko; Okudera, Toshimitsu; Watanabe, Taisuke; Isobe, Kazushige; Suzuki, Masashi; Masuki, Hideo; Okudera, Hajime; Uematsu, Kohya; Nakata, Koh; Kawase, Tomoyuki

2016-11-01

Platelet-rich plasma (PRP) is widely used in regenerative medicine because of its high concentrations of various growth factors and platelets. However, the distribution of blood cell components has not been investigated in either PRP or other PRP derivatives. In this study, we focused on plasma rich in growth factors (PRGF), a PRP derivative, and analyzed the distributions of platelets and white blood cells (WBCs). Peripheral blood samples were collected from healthy volunteers ( N  = 14) and centrifuged to prepare PRGF and PRP. Blood cells were counted using an automated hematology analyzer. The effects of PRP and PRGF preparations on cell proliferation were determined using human periosteal cells. In the PRGF preparations, both red blood cells and WBCs were almost completely eliminated, and platelets were concentrated by 2.84-fold, whereas in the PRP preparations, both platelets and WBCs were similarly concentrated by 8.79- and 5.51-fold, respectively. Platelet counts in the PRGF preparations were positively correlated with platelet counts in the whole blood samples, while the platelet concentration rate was negatively correlated with red blood cell counts in the whole blood samples. In contrast, platelet counts and concentration rates in the PRP preparations were significantly influenced by WBC counts in whole blood samples. The PRP preparations, but not the PRGF preparations, significantly suppressed cell growth at higher doses in vitro. Therefore, these results suggest that PRGF preparations can clearly be distinguished from PRP preparations by both inclusion of WBCs and dose-dependent stimulation of periosteal cell proliferation in vitro.

Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects

PubMed Central

Nishiyama, Kazuhiko; Okudera, Toshimitsu; Watanabe, Taisuke; Isobe, Kazushige; Suzuki, Masashi; Masuki, Hideo; Okudera, Hajime; Uematsu, Kohya; Nakata, Koh

2016-01-01

Abstract Platelet‐rich plasma (PRP) is widely used in regenerative medicine because of its high concentrations of various growth factors and platelets. However, the distribution of blood cell components has not been investigated in either PRP or other PRP derivatives. In this study, we focused on plasma rich in growth factors (PRGF), a PRP derivative, and analyzed the distributions of platelets and white blood cells (WBCs). Peripheral blood samples were collected from healthy volunteers (N = 14) and centrifuged to prepare PRGF and PRP. Blood cells were counted using an automated hematology analyzer. The effects of PRP and PRGF preparations on cell proliferation were determined using human periosteal cells. In the PRGF preparations, both red blood cells and WBCs were almost completely eliminated, and platelets were concentrated by 2.84‐fold, whereas in the PRP preparations, both platelets and WBCs were similarly concentrated by 8.79‐ and 5.51‐fold, respectively. Platelet counts in the PRGF preparations were positively correlated with platelet counts in the whole blood samples, while the platelet concentration rate was negatively correlated with red blood cell counts in the whole blood samples. In contrast, platelet counts and concentration rates in the PRP preparations were significantly influenced by WBC counts in whole blood samples. The PRP preparations, but not the PRGF preparations, significantly suppressed cell growth at higher doses in vitro. Therefore, these results suggest that PRGF preparations can clearly be distinguished from PRP preparations by both inclusion of WBCs and dose‐dependent stimulation of periosteal cell proliferation in vitro. PMID:29744155

Donor Cell Composition and Reactivity Predict Risk of Acute Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation.

PubMed

Sairafi, Darius; Stikvoort, Arwen; Gertow, Jens; Mattsson, Jonas; Uhlin, Michael

2016-01-01

Background . Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). We designed a functional assay for assessment of individual risk for acute GVHD. Study Design and Methods . Blood samples were collected from patients and donors before HSCT. Two groups of seven patients each were selected, one in which individuals developed acute GVHD grades II-IV and one in which none showed any clinical signs of GVHD. Peripheral blood mononuclear cells (PBMCs) isolated from donors were incubated in mixed lymphocyte cultures (MLCs) with recipient PBMCs. The cells were characterized by flow cytometry before and after MLC. Results . Samples from donors in the GVHD group contained significantly lower frequencies of naïve γδ T-cells and T-cells expressing NK-cell markers CD56 and CD94. Donor samples in this group also exhibited lower frequencies of naïve CD95 + T-cells compared to controls. After MLC, there were dissimilarities in the CD4/CD8 T-cell ratio and frequency of CD69 + T-cells between the two patient groups, with the non-GVHD group showing higher frequencies of CD8 + and CD69 + T-cells. Conclusion . We conclude that a thorough flow cytometric analysis of donor cells for phenotype and allogeneic reactivity may be of value when assessing pretransplant risk for severe acute GVHD.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures.

PubMed

Wiklander, Oscar P B; Bostancioglu, R Beklem; Welsh, Joshua A; Zickler, Antje M; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O; Mohammad, Dara K; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z; Jones, Jennifer C; El Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Effective cancer laser-therapy design through the integration of nanotechnology and computational treatment planning models

NASA Astrophysics Data System (ADS)

Fisher, Jessica W.; Rylander, Marissa Nichole

2008-02-01

Laser therapies can provide a minimally invasive treatment alternative to surgical resection of tumors. However, the effectiveness of these therapies is limited due to nonspecific heating of target tissue which often leads to healthy tissue injury and extended treatment durations. These therapies can be further compromised due to heat shock protein (HSP) induction in tumor regions where non-lethal temperature elevation occurs, thereby imparting enhanced tumor cell viability and resistance to subsequent chemotherapy and radiation treatments. Introducing multi-walled nanotubes (MWNT) into target tissue prior to laser irradiation increases heating selectivity permitting more precise thermal energy delivery to the tumor region and enhances thermal deposition thereby increasing tumor injury and reducing HSP expression induction. This study investigated the impact of MWNT inclusion in untreated and laser irradiated monolayer cell culture and cell phantom model. Cell viability remained high for all samples with MWNT inclusion and cells integrated into alginate phantoms, demonstrating the non-toxic nature of both MWNTs and alginate phantom models. Following, laser irradiation samples with MWNT inclusion exhibited dramatic temperature elevations and decreased cell viability compared to samples without MWNT. In the cell monolayer studies, laser irradiation of samples with MWNT inclusion experienced up-regulated HSP27, 70 and 90 expression as compared to laser only or untreated samples due to greater temperature increases albeit below the threshold for cell death. Further tuning of laser parameters will permit effective cell killing and down-regulation of HSP. Due to optimal tuning of laser parameters and inclusion of MWNT in phantom models, extensive temperature elevations and cell death occurred, demonstrating MWNT-mediated laser therapy as a viable therapy option when parameters are optimized. In conclusion, MWNT-mediated laser therapies show great promise for effective tumor destruction, but require determination of appropriate MWNT characteristics and laser parameters for maximum tumor destruction.

Distinct gene expression profiles determine molecular treatment response in childhood acute lymphoblastic leukemia.

PubMed

Cario, Gunnar; Stanulla, Martin; Fine, Bernard M; Teuffel, Oliver; Neuhoff, Nils V; Schrauder, André; Flohr, Thomas; Schäfer, Beat W; Bartram, Claus R; Welte, Karl; Schlegelberger, Brigitte; Schrappe, Martin

2005-01-15

Treatment resistance, as indicated by the presence of high levels of minimal residual disease (MRD) after induction therapy and induction consolidation, is associated with a poor prognosis in childhood acute lymphoblastic leukemia (ALL). We hypothesized that treatment resistance is an intrinsic feature of ALL cells reflected in the gene expression pattern and that resistance to chemotherapy can be predicted before treatment. To test these hypotheses, gene expression signatures of ALL samples with high MRD load were compared with those of samples without measurable MRD during treatment. We identified 54 genes that clearly distinguished resistant from sensitive ALL samples. Genes with low expression in resistant samples were predominantly associated with cell-cycle progression and apoptosis, suggesting that impaired cell proliferation and apoptosis are involved in treatment resistance. Prediction analysis using randomly selected samples as a training set and the remaining samples as a test set revealed an accuracy of 84%. We conclude that resistance to chemotherapy seems at least in part to be an intrinsic feature of ALL cells. Because treatment response could be predicted with high accuracy, gene expression profiling could become a clinically relevant tool for treatment stratification in the early course of childhood ALL.

Distinctive toxicity of TiO2 rutile/anatase mixed phase nanoparticles on Caco-2 cells.

PubMed

Gerloff, Kirsten; Fenoglio, Ivana; Carella, Emanuele; Kolling, Julia; Albrecht, Catrin; Boots, Agnes W; Förster, Irmgard; Schins, Roel P F

2012-03-19

Titanium dioxide has a long-standing use as a food additive. Micrometric powders are, e.g., applied as whiteners in confectionary or dairy products. Possible hazards of ingested nanometric TiO(2) particles for humans and the potential influence of varying specific surface area (SSA) are currently under discussion. Five TiO(2)-samples were analyzed for purity, crystallinity, primary particle size, SSA, ζ potential, and aggregation/agglomeration. Their potential to induce cytotoxicity, oxidative stress, and DNA damage was evaluated in human intestinal Caco-2 cells. Only anatase-rutile containing samples, in contrast to the pure anatase samples, induced significant LDH leakage or mild DNA damage (Fpg-comet assay). Evaluation of the metabolic competence of the cells (WST-1 assay) revealed a highly significant correlation between the SSA of the anatase samples and cytotoxicity. The anatase/rutile samples showed higher toxicity per unit surface area than the pure anatase powders. However, none of the samples affected cellular markers of oxidative stress. Our findings suggest that both SSA and crystallinity are critical determinants of TiO(2)-toxicity toward intestinal cells. © 2012 American Chemical Society

Effect of Pore Size and Porosity on the Biomechanical Properties and Cytocompatibility of Porous NiTi Alloys

PubMed Central

Jian, Yu-Tao; Yang, Yue; Tian, Tian; Stanford, Clark; Zhang, Xin-Ping; Zhao, Ke

2015-01-01

Five types of porous Nickel-Titanium (NiTi) alloy samples of different porosities and pore sizes were fabricated. According to compressive and fracture strengths, three groups of porous NiTi alloy samples underwent further cytocompatibility experiments. Porous NiTi alloys exhibited a lower Young’s modulus (2.0 GPa ~ 0.8 GPa). Both compressive strength (108.8 MPa ~ 56.2 MPa) and fracture strength (64.6 MPa ~ 41.6 MPa) decreased gradually with increasing mean pore size (MPS). Cells grew and spread well on all porous NiTi alloy samples. Cells attached more strongly on control group and blank group than on all porous NiTi alloy samples (p < 0.05). Cell adhesion on porous NiTi alloys was correlated negatively to MPS (277.2 μm ~ 566.5 μm; p < 0.05). More cells proliferated on control group and blank group than on all porous NiTi alloy samples (p < 0.05). Cellular ALP activity on all porous NiTi alloy samples was higher than on control group and blank group (p < 0.05). The porous NiTi alloys with optimized pore size could be a potential orthopedic material. PMID:26047515

Vibro-Acoustic Forecasts for STS (Space Transportation System) Launches at V23, Vandenberg AFB: Results Summary and the Payload Preparation Room

DTIC Science & Technology

1985-05-08

Displacement Time Series Forecasts for Channels 2 Through 8 and (b) Channels 9 Through 16 33 17. Sample PSD Plots for ( a ) Levels 99 and 119 East Cell Rail...for Sensors at ( a ) Levels 99 and 119 on the West Cell Rail, (b) Level 69 on the East and West Cell Rail Footings, and (c) Level 99 on the West Cell Rail...17. Sample PSD Plots for ( a ) Levels 99 and 119 East Cell Rail Locations, (b) level 69 Sensors on the East and West Cell Rail Footings, and (c) Level

Ammonium Sulfate Evaporites Associated With Uranium Mill Tailings Disposal Cells

NASA Astrophysics Data System (ADS)

Wendlandt, R. F.; Harrison, W. J.

2006-12-01

The waste products of uranium mill operations are complex and dependent on the ore mineralogy, milling process (e.g., low pH vs. high pH), and operational status of the mill among other things. The White Mesa Mill, Utah, was visited during both quiescent (July 2004) and operational phases (August 2005) to collect liquid and solid samples from the active evaporation and storage ponds environments (Cells 1 and 3). Cell 4, which was unused and being excavated at the times of both samplings, yielded solids accumulated through the history of that cell's use. Raffinate samples are concentrated Na-Mg-Al-Fe-SO4-NO3(-NH4) brines characterized by extreme enrichments in REE and transition elements. Ionic strengths, calculated using the Pitzer activity coefficient model varied from 25M (pH = 1 at 25°C) in Cell 1 and 12M (pH = 2.7) in Cell 3 during July 2004, to 5M (pH = 1.5) in Cell 1 and 1.2M (pH = 2.9) in Cell 3 during August 2005. At the first sampling, the dominant anion was sulfate in Cell 1 and nitrate in Cell 3. At the time of the second sampling, both cells were dominated by sulfate. During July 2004, there was significant evaporative drawdown in the ponds, resulting in 3 variably colored zones (~7m) of mineralogically complex evaporites at the cell margins. During August 2005, the operational nature of the mill and the addition of fresh water had produced high water levels in Cells 1 and 3. Evaporation crusts were recognized around the margins of the cells but they were <2m in extent. XRD analyses document the presence of boussingaultite, (NH4)2Mg(SO4)2.6H2O, which was actively precipitating from Cell 1 during 2004, tschermigite, (NH4)Al(SO4)2.12H2O, gypsum, and polymorphs of Na2SO4 including thenardite. ESEM imaging and EDS analyses of crusts reveal complex parageneses involving the above-mentioned phases and NH4-bearing metavoltine, K2Na6Fe^{+2}Fe6^{+3}(SO4)12O2.18H2O, among others. Ksp calculations and field relations are consistent with a precipitation sequence of tschermigite followed by boussingaultite and metavoltine.

Can an inadequate cervical cytology sample in ThinPrep be converted to a satisfactory sample by processing it with a SurePath preparation?

PubMed

Sørbye, Sveinung Wergeland; Pedersen, Mette Kristin; Ekeberg, Bente; Williams, Merete E Johansen; Sauer, Torill; Chen, Ying

2017-01-01

The Norwegian Cervical Cancer Screening Program recommends screening every 3 years for women between 25 and 69 years of age. There is a large difference in the percentage of unsatisfactory samples between laboratories that use different brands of liquid-based cytology. We wished to examine if inadequate ThinPrep samples could be satisfactory by processing them with the SurePath protocol. A total of 187 inadequate ThinPrep specimens from the Department of Clinical Pathology at University Hospital of North Norway were sent to Akershus University Hospital for conversion to SurePath medium. Ninety-one (48.7%) were processed through the automated "gynecologic" application for cervix cytology samples, and 96 (51.3%) were processed with the "nongynecological" automatic program. Out of 187 samples that had been unsatisfactory by ThinPrep, 93 (49.7%) were satisfactory after being converted to SurePath. The rate of satisfactory cytology was 36.6% and 62.5% for samples run through the "gynecology" program and "nongynecology" program, respectively. Of the 93 samples that became satisfactory after conversion from ThinPrep to SurePath, 80 (86.0%) were screened as normal while 13 samples (14.0%) were given an abnormal diagnosis, which included 5 atypical squamous cells of undetermined significance, 5 low-grade squamous intraepithelial lesion, 2 atypical glandular cells not otherwise specified, and 1 atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion. A total of 2.1% (4/187) of the women got a diagnosis of cervical intraepithelial neoplasia 2 or higher at a later follow-up. Converting cytology samples from ThinPrep to SurePath processing can reduce the number of unsatisfactory samples. The samples should be run through the "nongynecology" program to ensure an adequate number of cells.

Filter paper-assisted cell transfer (FaCT) technique: A novel cell-sampling technique for intraoperative diagnosis of central nervous system tumors.

PubMed

Kawamura, Jumpei; Kamoshida, Shingo; Shimakata, Takaaki; Hayashi, Yurie; Sakamaki, Kuniko; Denda, Tamami; Kawai, Kenji; Kuwao, Sadahito

2017-04-01

Intraoperative diagnosis of central nervous system (CNS) tumors provides critical guidance to surgeons in the determination of surgical resection margins and treatment. The techniques and preparations used for the intraoperative diagnosis of CNS tumors include frozen sectioning and cytologic methods (squash smear and touch imprint). Cytologic specimens, which do not have freezing artifacts, are important as an adjuvant tool to frozen sections. However, if the amount of submitted tissue samples is limited, then it is difficult to prepare both frozen sections and squash smears or touch imprint specimens from a single sample at the same time. Therefore, the objective of this study was to derive cells directly from filter paper on which tumor samples are placed. The authors established the filter paper-assisted cell transfer (FaCT) smear technique, in which tumor cells are transferred onto a glass slide directly from the filter paper sample spot after the biopsy is removed. Cell yields and diagnostic accuracy of the FaCT smears were assessed in 40 CNS tumors. FaCT smears had ample cell numbers and well preserved cell morphology sufficient for cytologic diagnosis, even if the submitted tissues were minimal. The overall diagnostic concordance rates between frozen sections and FaCT smears were 90% and 87.5%, respectively (no significant differences). When combining FaCT smears with frozen sections, the diagnostic concordance rate rose to 92.5%. The current results suggest that the FaCT smear technique is a simple and effective processing method that has significant value for intraoperative diagnosis of CNS tumors. Cancer Cytopathol 2017;125:277-282. © 2016 American Cancer Society. © 2017 American Cancer Society.

Stria vascularis and cochlear hair cell changes in syphilis: A human temporal bone study.

PubMed

Hızlı, Ömer; Kaya, Serdar; Hızlı, Pelin; Paparella, Michael M; Cureoglu, Sebahattin

2016-12-01

To observe any changes in stria vascularis and cochlear hair cells in patients with syphilis. We examined 13 human temporal bone samples from 8 patients with syphilis (our syphilis group), as well as 12 histopathologically normal samples from 9 age-matched patients without syphilis (our control group). We compared, between the two groups, the mean area of the stria vascularis (measured with conventional light microscopy connected to a personal computer) and the mean percentage of cochlear hair cell loss (obtained from cytocochleograms). In our syphilis group, only 1 (7.7%) of the 13 samples had precipitate in the endolymphatic or perilymphatic spaces; 8 (61.5%) of the samples revealed the presence of endolymphatic hydrops (4 cochlear, 4 saccular). The mean area of the stria vascularis did not significantly differ, in any turn of the cochlea, between the 2 groups (P>0.1). However, we did find significant differences between the 2 groups in the mean percentage of outer hair cells in the apical turn (P<0.026) and in the mean percentage of inner hair cells in the basal (P=0.001), middle (P=0.004), and apical (P=0.018) turns. In 7 samples in our syphilis group, we observed either complete loss of the organ of Corti or a flattened organ of Corti without any cells in addition to the absence of both outer and inner hair cells. In this study, syphilis led either to complete loss of the organ of Corti or to significant loss of cochlear hair cells, in addition to cochleosaccular hydrops. But the area of the stria vascularis did not change. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

In-vitro evaluation of Polylactic acid (PLA) manufactured by fused deposition modeling.

PubMed

Wurm, Matthias C; Möst, Tobias; Bergauer, Bastian; Rietzel, Dominik; Neukam, Friedrich Wilhelm; Cifuentes, Sandra C; Wilmowsky, Cornelius von

2017-01-01

With additive manufacturing (AM) individual and biocompatible implants can be generated by using suitable materials. The aim of this study was to investigate the biological effects of polylactic acid (PLA) manufactured by Fused Deposition Modeling (FDM) on osteoblasts in vitro according to European Norm / International Organization for Standardization 10,993-5. Human osteoblasts (hFOB 1.19) were seeded onto PLA samples produced by FDM and investigated for cell viability by fluorescence staining after 24 h. Cell proliferation was measured after 1, 3, 7 and 10 days by cell-counting and cell morphology was evaluated by scanning electron microscopy. For control, we used titanium samples and polystyrene (PS). Cell viability showed higher viability on PLA (95,3% ± 2.1%) than in control (91,7% ±2,7%). Cell proliferation was highest in the control group (polystyrene) and higher on PLA samples compared to the titanium samples. Scanning electron microscopy revealed homogenous covering of sample surface with regularly spread cells on PLA as well as on titanium. The manufacturing of PLA discs from polylactic acid using FDM was successful. The in vitro investigation with human fetal osteoblasts showed no cytotoxic effects. Furthermore, FDM does not seem to alter biocompatibility of PLA. Nonetheless osteoblasts showed reduced growth on PLA compared to the polystyrene control within the cell experiments. This could be attributed to surface roughness and possible release of residual monomers. Those influences could be investigated in further studies and thus lead to improvement in the additive manufacturing process. In addition, further research focused on the effect of PLA on bone growth should follow. In summary, PLA processed in Fused Deposition Modelling seems to be an attractive material and method for reconstructive surgery because of their biocompatibility and the possibility to produce individually shaped scaffolds.

Human respiratory syncytial virus load normalized by cell quantification as predictor of acute respiratory tract infection.

PubMed

Gómez-Novo, Miriam; Boga, José A; Álvarez-Argüelles, Marta E; Rojo-Alba, Susana; Fernández, Ana; Menéndez, María J; de Oña, María; Melón, Santiago

2018-05-01

Human respiratory syncytial virus (HRSV) is a common cause of respiratory infections. The main objective is to analyze the prediction ability of viral load of HRSV normalized by cell number in respiratory symptoms. A prospective, descriptive, and analytical study was performed. From 7307 respiratory samples processed between December 2014 to April 2016, 1019 HRSV-positive samples, were included in this study. Low respiratory tract infection was present in 729 patients (71.54%). Normalized HRSV load was calculated by quantification of HRSV genome and human β-globin gene and expressed as log10 copies/1000 cells. HRSV mean loads were 4.09 ± 2.08 and 4.82 ± 2.09 log10 copies/1000 cells in the 549 pharyngeal and 470 nasopharyngeal samples, respectively (P < 0.001). The viral mean load was 4.81 ± 1.98 log10 copies/1000 cells for patients under the age of 4-year-old (P < 0.001). The viral mean loads were 4.51 ± 2.04 cells in patients with low respiratory tract infection and 4.22 ± 2.28 log10 copies/1000 cells with upper respiratory tract infection or febrile syndrome (P < 0.05). A possible cut off value to predict LRTI evolution was tentatively established. Normalization of viral load by cell number in the samples is essential to ensure an optimal virological molecular diagnosis avoiding that the quality of samples affects the results. A high viral load can be a useful marker to predict disease progression. © 2018 Wiley Periodicals, Inc.

Stria Vascularis and Cochlear Hair Cell Changes in Syphilis: A Human Temporal Bone Study

PubMed Central

Hızlı, Ömer; Kaya, Serdar; Hızlı, Pelin; Paparella, Michael M.; Cureoglu, Sebahattin

2016-01-01

Objective To observe any changes in stria vascularis and cochlear hair cells in patients with syphilis Materials and Methods We examined 13 human temporal bone samples from 8 patients with syphilis (our syphilis group), as well as 12 histopathologically normal samples from 9 age-matched patients without syphilis (our control group). We compared, between the 2 groups, the mean area of the stria vascularis (measured with conventional light microscopy connected to a personal computer) and the mean percentage of cochlear hair cell loss (obtained from cytocochleograms). Results In our syphilis group, only 1 (7.7%) of the 13 samples had precipitate in the endolymphatic or perilymphatic spaces; 8 (61.5%) of the samples revealed the presence of endolymphatic hydrops (4 cochlear, 4 saccular). The mean area of the stria vascularis area did not significantly differ, in any turn of the cochlea, between the 2 groups (P > 0.1). However, we did find significant differences between the 2 groups in the mean percentage of outer hair cells in the apical turn (P < 0.026) and in the mean percentage of inner hair cells in the basal (P = 0.001), middle (P = 0.004), and apical (P = 0.018) turns. In 7 samples in our syphilis group, we observed either complete loss of the organ of Corti or a flattened organ of Corti without any cells in addition to the absence of both outer and inner hair cells. Conclusion In this study, syphilis led either to complete loss of the organ of Corti or to significant loss of cochlear hair cells, in addition to cochleosaccular hydrops. But the area of the stria vascularis did not change. PMID:26860231

Preparation and Loading Process of Single Crystalline Samples into a Gas Environmental Cell Holder for In Situ Atomic Resolution Scanning Transmission Electron Microscopic Observation.

PubMed

Straubinger, Rainer; Beyer, Andreas; Volz, Kerstin

2016-06-01

A reproducible way to transfer a single crystalline sample into a gas environmental cell holder for in situ transmission electron microscopic (TEM) analysis is shown in this study. As in situ holders have only single-tilt capability, it is necessary to prepare the sample precisely along a specific zone axis. This can be achieved by a very accurate focused ion beam lift-out preparation. We show a step-by-step procedure to prepare the sample and transfer it into the gas environmental cell. The sample material is a GaP/Ga(NAsP)/GaP multi-quantum well structure on Si. Scanning TEM observations prove that it is possible to achieve atomic resolution at very high temperatures in a nitrogen environment of 100,000 Pa.

Comparison of hematologic values in blood samples with lithium heparin or dipotassium ethylenediaminetetraacetic acid anticoagulants in Hispaniolan Amazon parrots (Amazona ventralis).

PubMed

Guzman, David Sanchez-Migallon; Mitchell, Mark A; Gaunt, Stephen D; Beaufrère, Hugues; Tully, Thomas N

2008-06-01

Blood samples were collected from 20 Hispaniolan Amazon parrots (Amazona ventralis) and were divided into tubes that contained dipotassium ethylenediaminetetraacetic acid (K2EDTA) and lithium heparin. Complete blood cell counts were determined in each sample within 2 hours of collection. The level of agreement in results was moderate for plasma protein, packed cell volume (PCV), and leukocyte, monocyte, and lymphocyte counts between the anticoagulants. Plasma protein and PCV values were significantly lower in samples with lithium heparin than in those with K2EDTA, whereas lymphocyte numbers were significantly higher in lithium heparin samples than in K2EDTA samples. The level of agreement was good for the other cell types (heterophils, eosinophils, and basophils) when comparing the different anticoagulants. The poor level of agreement between anticoagulants with the increase in thrombocyte clumping in lithium heparin samples indicates that the use of lithium heparin as anticoagulant may affect thrombocyte count. No negative effects on morphology and staining of blood cells were apparent in smears from heparin samples compared with K2EDTA samples. Within the different values compared, the limits of agreement are small enough to be confident that lithium heparin can be used for routine CBC counts in a clinical setting. The use of the same anticoagulant should be recommended to follow trends within the same patient, especially when considering plasma protein concentration, PCV, and lymphocyte count.

Fully Integrated Microfluidic Device for Direct Sample-to-Answer Genetic Analysis

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Grodzinski, Piotr

Integration of microfluidics technology with DNA microarrays enables building complete sample-to-answer systems that are useful in many applications such as clinic diagnostics. In this chapter, a fully integrated microfluidic device [1] that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and a DNA microarray sensor to perform DNA analysis of complex biological sample solutions is present. This device can perform on-chip sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis) of complex biological sample solutions (such as whole blood), polymerase chain reaction, DNA hybridization, and electrochemical detection. A few novel microfluidic techniques were developed and employed. A micromix-ing technique based on a cavitation microstreaming principle was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads. This technique was also employed to accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from ~mL whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

PubMed

Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

2012-11-01

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

Evaluation of a neonatal screening program for sickle-cell disease.

PubMed

Eller, Rodrigo; da Silva, Denise Bousfield

2016-01-01

Evaluate the Neonatal Screening Program of the Health Secretariat of the State of Santa Catarina for sickle-cell disease, from January 2003 to December 2012, regarding program coverage and disease frequency. Descriptive, observational, cross-sectional study with retrospective data collection. The variables analyzed were: number of live births in the State of Santa Catarina; number of screened children; number of children diagnosed with sickle-cell trait and sickle-cell disease; type of sickle-cell disease diagnosed; age at the time of sample collection, ethnicity/skin color, gender, and origin of children with sickle-cell disease. Descriptive measures and frequency tables were used for data analysis. During the study period, there were 848,833 live births and 730,412 samples were screened by the program, resulting in a coverage of 86.0%. There were 6173 samples positive for sickle-cell trait and 39 for sickle-cell disease. Among children with sickle-cell disease, the median age at the time of sample collection was 6 days. Regarding the ethnicity/skin color, 25 (64.1%) children were white, seven were black, and seven others were not specified. The Midwest and the Highland (Planalto Serrano) of Santa Catarina were the regions with the highest incidence of sickle-cell disease. Coverage by the Neonatal Screening Program of Santa Catarina is good, but did not demonstrate an improvement trend over the years. The frequency of sickle-cell disease is low and lower than in the North, Northeast, and Midwest regions. The median age in days at the time of collection is older than the age recommended by the Ministry of Health. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Evaluation of commercial kit based on loop-mediated isothermal amplification for rapid detection of low levels of uninjured and injured Salmonella on duck meat, bean sprouts, and fishballs in Singapore.

PubMed

Lim, Hazel Sin Yue; Zheng, Qianwang; Miks-Krajnik, Marta; Turner, Matthew; Yuk, Hyun-Gyun

2015-06-01

The objective of this study was to evaluate performance of the commercial kit based on loop-mediated isothermal amplification (LAMP) in comparison with the International Organization for Standardization method for detecting uninjured and sublethally injured Salmonella cells artificially inoculated at levels of 10(0) and 10(1) CFU/25 g on raw duck wing, raw mung bean sprouts, and processed fishballs. Injured cells were prepared by a heat treatment for duck wings and fishball samples and a chlorine treatment for bean sprout samples. Additionally, a validation study was performed on naturally contaminated food samples sold in Singapore. A total of 110 samples of each commodity were analyzed in this study. Regardless of inoculum levels, the detection by the commercial LAMP kit showed 100% sensitivity and specificity for both inoculated and uninoculated samples compared with the International Organization for Standardization method, with the exception of bean sprout samples. Only 20% of bean sprout samples inoculated with 10(0) CFU/25 g injured Salmonella cells were positive by using the commercial LAMP-based kit. However, all negative samples became positive following a secondary enrichment in Rappaport-Vassiliadis medium with soy broth or after concentration by centrifugation. These results suggest that secondary enrichment or centrifugation should be considered as an additional step to increase the sensitivity of the commercial LAMP-based kit with low numbers of injured target cells in samples with high background microflora (such as mung bean sprouts). The validation study also showed that the commercial LAMP-based kit provided 91% sensitivity and 95% specificity for naturally contaminated samples. Thus, this study demonstrates that the commercial LAMP-based kit might be a cost-effective method, as this system could provide rapid, accurate detection of both uninjured and injured Salmonella cells on raw duck wings, raw mung bean sprouts, and processed fishballs in less than 26 h.

Ultraviolet micro-Raman spectrograph for the detection of small numbers of bacterial cells

NASA Astrophysics Data System (ADS)

Chadha, S.; Nelson, W. H.; Sperry, J. F.

1993-11-01

The construction of a practical UV micro-Raman spectrograph capable of selective excitation of bacterial cells and other microscopic samples has been described. A reflective objective is used to focus cw laser light on a sample and at the same time collect the scattered light at 180°. With the aid of a quartz lens the image produced is focused on the slits of a spectrograph equipped with a single 2400 grooves/mm grating optimized for 250 nm. Spectra were detected by means of a blue-intensified diode array detector. Resonance Raman spectra of Bacillus subtilis and Flavobacterium capsulatum excited by the 257.2 nm output of a cw laser were recorded in the 900-1800 cm-1 region. Bacterial cells were immobilized on a quartz plate by means of polylysine and were counted visually. Cooling was required to retard sample degradation. Sample sizes ranged from 1 to 50 cells with excitation times varying from 15 to 180 s. Excellent spectra have been obtained from 20 cells in 15 s using a spectrograph having only 3% throughput.

Method and apparatus for biological material separation

DOEpatents

Robinson, Donna L.

2005-05-10

There has been invented an apparatus comprising a separation barrier for excluding denser cell materials from less dense cell materials after centrifuging of the cells so that selected materials can be withdrawn from the less dense cell materials without inclusion of the denser cell materials or clogging of sampling equipment with denser cell materials. Cells from which selected material is to be withdrawn are centrifuged, either as cells or cells in media. Once the denser cell materials are isolated in a layer by centrifugal force, an invention screen or seive is submerged in the less dense cell material to a level above the layer of denser cell materials to isolate the denser cell materials from the less dense cell materials, preventing mixing of the denser cell materials back into the less dense cell materials when the cells or the cells in media are no longer being centrifuged and to prevent clogging of sampling equipment with denser cell materials. In a particularly useful application of the invention method and apparatus, plasmid DNA can be withdrawn from less dense cell materials without contamination or interference with denser cell materials.

Differentiating quiescent cancer cell populations in heterogeneous samples with fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Heaster, Tiffany M.; Walsh, Alex J.; Skala, Melissa C.

2016-03-01

Measurement of relative fluorescence intensities of NAD(P)H and FAD with fluorescence lifetime imaging (FLIM) allows metabolic characterization of cancerous populations and correlation to treatment response. However, quiescent populations of cancer cells introduce heterogeneity to the tumor and exhibit resistance to standard therapies, requiring a better understanding of this influence on treatment outcome. Significant differences were observed between proliferating and quiescent cell populations upon comparison of respective redox ratios (p<0.05) and FAD lifetimes (p<0.05) across monolayers and in mixed samples. These results demonstrate that metabolic activity may function as a marker for separation and characterization of proliferating and quiescent cancer cells within mixed samples, contributing to comprehensive investigation of heterogeneity-dependent drug resistance.

Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells.

PubMed

Wojcik, Michal; Hauser, Margaret; Li, Wan; Moon, Seonah; Xu, Ke

2015-06-11

The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.

Real-time and accelerated outdoor endurance testing of solar cells

NASA Technical Reports Server (NTRS)

Forestieri, A. F.; Anagnostou, E.

1977-01-01

Real-time and accelerated outdoor endurance testing was performed on a variety of samples of interest to the National Photovoltaic Conversion Program. The real-time tests were performed at seven different sites and the accelerated tests were performed at one of those sites in the southwestern United States. The purpose of the tests were to help evaluate the lifetime of photovoltaic systems. Three types of samples were tested; transmission samples of possible cover materials, sub-modules constructed using these materials attached to solar cells, and solar cell modules produced by the manufacturers for the ERDA program. Results indicate that suitable cover materials are glass, FEP-A and PFA. Dirt accumulation and cleanability are important factors in the selection of solar cell module covers and encapsulants.

PD-1+Tim-3+ CD8+ T Lymphocytes Display Varied Degrees of Functional Exhaustion in Patients with Regionally Metastatic Differentiated Thyroid Cancer

PubMed Central

Severson, Jill J.; Serracino, Hilary S.; Mateescu, Valerica; Raeburn, Christopher D.; C.McIntyre, Robert; Sams, Sharon B.; Haugen, Bryan R.; French, Jena D.

2015-01-01

Regional metastatic differentiated thyroid cancer (mDTC) provides a unique model in which to study the tumor-immune interface. These lymph node (LN) metastases persist for years, generally without progression to distant metastases. While the immune system likely impedes disease progression, it is unsuccessful in eliminating disease. Our previous studies revealed that programmed death-1 (PD-1)+ T cells were enriched in tumor-involved lymph nodes (TILN). Tumor-associated leukocytes and tumor cells were collected from grossly involved LNs from 12 patients to further characterize the phenotype and functional potential of mDTC-associated PD-1+ T cells. PD-1+CD4+ and PD-1+CD8+ T cells were enriched in 8/12 TILN samples. PD-1+ T cells co-expressed Tim-3 and CD69 and failed to down-regulate CD27. CD8+ T cells, but not CD4+ T cells, from these samples were variably deficient in their ability to produce effector cytokines when compared to control TILNs that lacked resident PD-1+ T cells. PD-1+CD8+ T cells were capable of exocytosis but lacked intracellular perforin. Surprisingly, T-cell proliferative capacity was largely maintained in all samples. Thus, while PD-1 expression by mDTC-associated CD8+ T cells was associated with dysfunction, exhaustion was not complete. Notably, molecular markers of exhaustion did not translate to dysfunction in all samples or in CD4+ T cells. Regulatory T (Treg) cells, PD-L1, and galectin-9 were commonly found in mDTC and likely contributed to the initiation of T-cell exhaustion and disease progression. Therapies that release the effects of PD-1 and Tim-3 and reduce the suppressive effects of Tregs may encourage tumor elimination in patients with mDTC. PMID:25701326

Prevalence of human cell material: DNA and RNA profiling of public and private objects and after activity scenarios.

PubMed

van den Berge, M; Ozcanhan, G; Zijlstra, S; Lindenbergh, A; Sijen, T

2016-03-01

Especially when minute evidentiary traces are analysed, background cell material unrelated to the crime may contribute to detectable levels in the genetic analyses. To gain understanding on the composition of human cell material residing on surfaces contributing to background traces, we performed DNA and mRNA profiling on samplings of various items. Samples were selected by considering events contributing to cell material deposits in exemplary activities (e.g. dragging a person by the trouser ankles), and can be grouped as public objects, private samples, transfer-related samples and washing machine experiments. Results show that high DNA yields do not necessarily relate to an increased number of contributors or to the detection of other cell types than skin. Background cellular material may be found on any type of public or private item. When a major contributor can be deduced in DNA profiles from private items, this can be a different person than the owner of the item. Also when a specific activity is performed and the areas of physical contact are analysed, the "perpetrator" does not necessarily represent the major contributor in the STR profile. Washing machine experiments show that transfer and persistence during laundry is limited for DNA and cell type dependent for RNA. Skin conditions such as the presence of sebum or sweat can promote DNA transfer. Results of this study, which encompasses 549 samples, increase our understanding regarding the prevalence of human cell material in background and activity scenarios. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Effect of high pressure treatment on liquid whole egg

NASA Astrophysics Data System (ADS)

Németh, Csaba; Dalmadi, István; Mráz, Balázs; Friedrich, László; Zeke, Ildikó; Juhász, Réka; Suhajda, Ágnes; Balla, Csaba

2012-06-01

In our tests, we artificially infected liquid whole egg samples with Salmonella enteritidis, Listeria monocytogenes, and Staphylococcus aureus bacteria, and then treated the samples in "Food Lab900" high hydrostatic pressure (HHP) instrument for 3-17 min at 200-400 MPa. Subsequently, the change of the viable cell count of the specific bacteria has been tested. In addition to the samples infected with various bacteria, non-infected samples were also treated in each test and the change in viable cell count, colour and viscosity of the samples upon the effect of the treatment. In summary, it can be concluded that in each test of our investigations, the viable cell count of S. enteritidis critical for egg products is reduced significantly, while the reduction of the total viable cell count was around two magnitudes. Additionally, based on our results, microbial destruction, reduction of enthalpy (denaturation of egg white) caused by the treatment at HPP, and colour change are primarily affected by the pressure level, while the changes in rheological properties are also significantly affected by the duration of high pressure treatment (p<0.05).

Persistent complement activation on tumor cells in breast cancer.

PubMed Central

Niculescu, F.; Rus, H. G.; Retegan, M.; Vlaicu, R.

1992-01-01

The neoantigens of the C5b-9 complement complex, IgG, C3, C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenetic effects in breast cancer. Images Figure 1 PMID:1374587

Method and automated apparatus for detecting coliform organisms

NASA Technical Reports Server (NTRS)

Dill, W. P.; Taylor, R. E.; Jeffers, E. L. (Inventor)

1980-01-01

Method and automated apparatus are disclosed for determining the time of detection of metabolically produced hydrogen by coliform bacteria cultured in an electroanalytical cell from the time the cell is inoculated with the bacteria. The detection time data provides bacteria concentration values. The apparatus is sequenced and controlled by a digital computer to discharge a spent sample, clean and sterilize the culture cell, provide a bacteria nutrient into the cell, control the temperature of the nutrient, inoculate the nutrient with a bacteria sample, measures the electrical potential difference produced by the cell, and measures the time of detection from inoculation.

Chip-based device for parallel sorting, amplification, detection, and identification of nucleic acid subsequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beer, Neil Reginald; Colston, Jr, Billy W.

An apparatus for chip-based sorting, amplification, detection, and identification of a sample having a planar substrate. The planar substrate is divided into cells. The cells are arranged on the planar substrate in rows and columns. Electrodes are located in the cells. A micro-reactor maker produces micro-reactors containing the sample. The micro-reactor maker is positioned to deliver the micro-reactors to the planar substrate. A microprocessor is connected to the electrodes for manipulating the micro-reactors on the planar substrate. A detector is positioned to interrogate the sample contained in the micro-reactors.

[Development of cell content and shedding of Prototheca spp. in milk from infected udder quarters of cows].

PubMed

Tenhagen, B A; Hille, A; Schmidt, A; Heuwieser, W

2005-02-01

It was the objective of this study to analyse shedding patterns and somatic cell counts in cows and quarters infected with Prototheca spp. and to evaluate two approaches to identify infected animals by somatic cell count (SCC) or by bacteriological analysis of pooled milk samples. Five lactating dairy cows, chronically infected with Prototheca spp. in at least one quarter were studied over 11 weeks to 13 months. Quarter milk samples and a pooled milk sample from 4 quarters were collected aseptically from all quarters of the cows on a weekly basis. Culture results of quarter milk and pooled samples were compared using cross tabulation. SCC of quarter milk samples and of pooled samples were related to the probability of detection in the infected quarters and cows, respectively. Shedding of Prototheca spp. was continuous in 2 of 8 quarters. In the other quarters negative samples were obtained sporadically or over a longer period (1 quarter). Overall, Prototheca spp. were isolated from 83.6% of quarter milk samples and 77.0% of pooled milk samples of infected quarters and cows. Somatic cell counts were higher in those samples from infected quarters that contained the algae than in negative samples (p < 0.0001). The same applied for composite samples from infected cows. Positive samples had higher SCC than negative samples. However, Prototheca spp. were also isolated from quarter milk and pooled samples with physiological SCC (i.e. < 10(5)/ml). Infected quarters that were dried off did not develop acute mastitis. However, drying off had no effect on the infection, i.e. samples collected at calving or 8 weeks after dry off still contained Prototheca spp. Results indicate that pre-selection of cows to be sampled for Prototheca spp. by SCC and the use of composite samples are probably inadequate in attempts to eradicate the disease. However, due to intermittent shedding of the algae in some cows, single herd sampling using quarter milk samples probably also fails to detect all infected cases. Therefore, continuous monitoring of problem cows with clinical mastitis or increased SCC in herds during eradication programs is recommended.

Whole blood analysis rotor assembly having removable cellular sedimentation bowl

DOEpatents

Burtis, C.A.; Johnson, W.F.

1975-08-26

A rotor assembly for performing photometric analyses using whole blood samples is described. Following static loading of a gross blood sample within a centrally located, removable, cell sedimentation bowl, the red blood cells in the gross sample are centrifugally separated from the plasma, the plasm displaced from the sedimentation bowl, and measured subvolumes of plasma distributed to respective sample analysis cuvettes positioned in an annular array about the rotor periphery. Means for adding reagents to the respective cuvettes are also described. (auth)

Development of Technologies for Early Detection and Stratification of Breast Cancer

DTIC Science & Technology

2014-10-01

cancer. Similar screening has been done in urine samples with CYR61 and LCN2 (Figure 2a-b). The Moses lab provided breast cancer urine samples and two ...diseased samples, while CYR61 appears to have two different populations whereas diseased urine samples have lower levels of the protein present. More... system for eDAR so that each isolated cell can be deposited into a well of a 96-well plate for high-throughput downstream single-cell digital

Control of intracellular ionic concentrations by mid-infrared laser irradiation

NASA Astrophysics Data System (ADS)

Takebe, G.; Yamauchi, T.; Shimizu, Y.; Dougakiuchi, T.

2018-02-01

We successfully induced intracellular ion concentration changes in live culture cells using mid-infrared laser irradiation. The laser used for irradiation was a quantum cascade laser with a wavelength of 6.1 micrometers. We tuned the power of the laser to be between 30 to 60 mW at the sample. Cell lines, namely HeLa and Chinese hamster ovary cell lines, were used. They were cultured on specially fabricated silicon-bottom dishes. Live cells were stained using ion-sensitive dyes such as Calcium Green-1. The mid-infrared light was incident on the cell samples from the bottom of the dish through the silicon plate, and fluorescence imaging of the ion concentrations was performed using an upright fluorescence microscope placed on top of the sample stage. The mid-infrared lasers were operated in the continuous wave mode and light irradiations onto the cells were temporally controlled using a mechanical shutter in a periodical on-and-off pattern in the second timescale. The cells showed oscillations in their ionic concentration, which was synchronized with the periodical mid-infrared irradiation, and the threshold power needed for evoking the ion concentration change was dependent on the cell types and ion species. These results demonstrated that mid-infrared light directly changed the ionic response within cells and had the ability to change cell functions.

Stem-Like Cell Characteristics from Breast Milk of Mothers with Preterm Infants as Compared to Mothers with Term Infants.

PubMed

Briere, Carrie-Ellen; Jensen, Todd; McGrath, Jacqueline M; Young, Erin E; Finck, Christine

2017-04-01

Breast milk stem cells are hypothesized to be involved in infant health and development. Our research team is the first known team to enroll mothers of hospitalized preterm infants during the first few weeks of lactation and compare stem cell phenotypes and gene expression to mothers of healthy full-term infants. Participants were recruited from a Level IV Neonatal Intensive Care Unit (preterm dyads) and the community (full-term dyads) in the northeastern United States. Mothers of hospitalized preterm infants (<37 weeks gestational age at birth) and mothers of healthy full-term infants (>39 weeks gestational age at birth). Breast milk stem-like cell populations were identified in both preterm and full-term breast milk samples. The data suggest variability in the proportion of stem cell phenotypes present, as well as statistically significant differential expression (both over- and underexpression) of stem cell-specific genetic markers when comparing mothers' milk for preterm and full-term births. Our findings indicate that (1) stem cells are present in preterm breast milk; (2) differential expression of stem cell-specific markers can be detected in preterm and full-term breast milk samples; and (3) the percentage of cells expressing the various stem cell-specific markers differs when preterm and full-term breast milk samples are compared.

Effect of graphene oxide ratio on the cell adhesion and growth behavior on a graphene oxide-coated silicon substrate

NASA Astrophysics Data System (ADS)

Jeong, Jin-Tak; Choi, Mun-Ki; Sim, Yumin; Lim, Jung-Taek; Kim, Gil-Sung; Seong, Maeng-Je; Hyung, Jung-Hwan; Kim, Keun Soo; Umar, Ahmad; Lee, Sang-Kwon

2016-09-01

Control of living cells on biocompatible materials or on modified substrates is important for the development of bio-applications, including biosensors and implant biomaterials. The topography and hydrophobicity of substrates highly affect cell adhesion, growth, and cell growth kinetics, which is of great importance in bio-applications. Herein, we investigate the adhesion, growth, and morphology of cultured breast cancer cells on a silicon substrate, on which graphene oxides (GO) was partially formed. By minimizing the size and amount of the GO-containing solution and the further annealing process, GO-coated Si samples were prepared which partially covered the Si substrates. The coverage of GO on Si samples decreases upon annealing. The behaviors of cells cultured on two samples have been observed, i.e. partially GO-coated Si (P-GO) and annealed partially GO-coated Si (Annealed p-GO), with a different coverage of GO. Indeed, the spreading area covered by the cells and the number of cells for a given culture period in the incubator were highly dependent on the hydrophobicity and the presence of oxygenated groups on GO and Si substrates, suggesting hydrophobicity-driven cell growth. Thus, the presented method can be used to control the cell growth via an appropriate surface modification.

Using single cell sequencing data to model the evolutionary history of a tumor.

PubMed

Kim, Kyung In; Simon, Richard

2014-01-24

The introduction of next-generation sequencing (NGS) technology has made it possible to detect genomic alterations within tumor cells on a large scale. However, most applications of NGS show the genetic content of mixtures of cells. Recently developed single cell sequencing technology can identify variation within a single cell. Characterization of multiple samples from a tumor using single cell sequencing can potentially provide information on the evolutionary history of that tumor. This may facilitate understanding how key mutations accumulate and evolve in lineages to form a heterogeneous tumor. We provide a computational method to infer an evolutionary mutation tree based on single cell sequencing data. Our approach differs from traditional phylogenetic tree approaches in that our mutation tree directly describes temporal order relationships among mutation sites. Our method also accommodates sequencing errors. Furthermore, we provide a method for estimating the proportion of time from the earliest mutation event of the sample to the most recent common ancestor of the sample of cells. Finally, we discuss current limitations on modeling with single cell sequencing data and possible improvements under those limitations. Inferring the temporal ordering of mutational sites using current single cell sequencing data is a challenge. Our proposed method may help elucidate relationships among key mutations and their role in tumor progression.

Cryopreservation of human whole blood allows immunophenotyping by flow cytometry up to 30days after cell isolation.

PubMed

Paredes, R Madelaine; Tadaki, Douglas K; Sooter, Amanda; Gamboni, Fabia; Sheppard, Forest

2018-01-01

Immunophenotyping of whole blood (WB) by flow cytometry (FC) is used clinically to assess a patient's immune status and also in biomedical research. Current protocols recommend storage of immunolabeled samples at 4°C with FC analysis to be completed within seven days. This data acquisition window can be extended to up to one year post-labeling, but this requires cryopreservation of the samples at ultra-low temperatures (≤-80°C or in liquid nitrogen). In this study we optimized a standardized cryopreservation protocol to enable preservation of immunolabeled, human WB samples at -20°C for FC and tested its effectiveness after 0, 5, 15 or 30days. Analysis of stored samples shows that this protocol effectively preserves immunolabeled WB samples and that the duration of storage has no effect on morphology, viability or frequency of WB cell subpopulations, and that the intensity of fluorescent signal from labeled extracellular markers is fully preserved for at least 15days, and up to 30days for some markers. We demonstrate that using this protocol, we are able to differentiate resting versus activated WB cells as demonstrated by detection of significantly increased expression of CD11b by myeloid cells in WB samples pretreated with LPS (100μg/mL for 12h). Finally, we show that this method allows for labeling and detection of the intracellular cytokine (IL-8) up to 30days following cryopreservation from myeloid cells, in previously labeled and cryopreserved WB samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells.

PubMed

Zhu, Ying; Piehowski, Paul D; Zhao, Rui; Chen, Jing; Shen, Yufeng; Moore, Ronald J; Shukla, Anil K; Petyuk, Vladislav A; Campbell-Thompson, Martha; Mathews, Clayton E; Smith, Richard D; Qian, Wei-Jun; Kelly, Ryan T

2018-02-28

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Ying; Piehowski, Paul D.; Zhao, Rui

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

Bloom of Cochlodinium polykrikoides (Dinophyceae: Gymnodiniales) in Bahía de La Paz, Gulf of California.

PubMed

Gárate-Lizárraga, Ismael

2013-02-15

As part of a continuing toxic microalgae monitoring program, phytoplankton samples were collected on 16-17 August 2012 at several sampling sites in the southern part of the Bahía de La Paz. A bloom of the dinoflagellate Cochlodinium polykrikoides was detected. Abundance of C. polykrikoides ranged from 73 to 276×10(3) cells L(-1) on the first day to 980-1425×10(3) cells L(-1) on the second day. Study of live specimens showed great variation in cell size and form, mainly as single cells or chains of two cells. Live cells were 30-47 μm long and 20-35 μm wide (n=30). Seawater temperature during the bloom was 29-30°C. Low densities of Cochlodinium convolutum, Cochlodinium helicoides, and Cochlodinium shuettii were also found in the samples of the bloom event. These high densities of C. polykrikoides did not lead to fish die-offs in the bay. Copyright © 2012 Elsevier Ltd. All rights reserved.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

DOE PAGES

Zhu, Ying; Piehowski, Paul D.; Zhao, Rui; ...

2018-02-28

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

PubMed

Monsanto, Megan M; White, Kevin S; Kim, Taeyong; Wang, Bingyan J; Fisher, Kristina; Ilves, Kelli; Khalafalla, Farid G; Casillas, Alexandria; Broughton, Kathleen; Mohsin, Sadia; Dembitsky, Walter P; Sussman, Mark A

2017-07-07

The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm 3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit + cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit - mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit + population is further enriched by selection for a CD133 + endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients. © 2017 American Heart Association, Inc.

Guided transect sampling - a new design combining prior information and field surveying

Treesearch

Anna Ringvall; Goran Stahl; Tomas Lamas

2000-01-01

Guided transect sampling is a two-stage sampling design in which prior information is used to guide the field survey in the second stage. In the first stage, broad strips are randomly selected and divided into grid-cells. For each cell a covariate value is estimated from remote sensing data, for example. The covariate is the basis for subsampling of a transect through...

Distilled single-cell genome sequencing and de novo assembly for sparse microbial communities.

PubMed

Taghavi, Zeinab; Movahedi, Narjes S; Draghici, Sorin; Chitsaz, Hamidreza

2013-10-01

Identification of every single genome present in a microbial sample is an important and challenging task with crucial applications. It is challenging because there are typically millions of cells in a microbial sample, the vast majority of which elude cultivation. The most accurate method to date is exhaustive single-cell sequencing using multiple displacement amplification, which is simply intractable for a large number of cells. However, there is hope for breaking this barrier, as the number of different cell types with distinct genome sequences is usually much smaller than the number of cells. Here, we present a novel divide and conquer method to sequence and de novo assemble all distinct genomes present in a microbial sample with a sequencing cost and computational complexity proportional to the number of genome types, rather than the number of cells. The method is implemented in a tool called Squeezambler. We evaluated Squeezambler on simulated data. The proposed divide and conquer method successfully reduces the cost of sequencing in comparison with the naïve exhaustive approach. Squeezambler and datasets are available at http://compbio.cs.wayne.edu/software/squeezambler/.

Optical diffraction tomography using a digital micromirror device for stable measurements of 4D refractive index tomography of cells

NASA Astrophysics Data System (ADS)

Shin, Seungwoo; Kim, Kyoohyun; Kim, Taeho; Yoon, Jonghee; Hong, Kihyun; Park, Jinah; Park, YongKeun

2016-03-01

Optical diffraction tomography (ODT) is an interferometric microscopy technique capable of measuring 3-D refractive index (RI) distribution of transparent samples. Multiple 2-D holograms of a sample illuminated with various angles are measured, from which 3-D RI map of the sample is reconstructed via the diffraction theory. ODT has been proved as a powerful tool for the study of biological cells, due to its non-invasiveness, label-free and quantitative imaging capability. Recently, our group has demonstrated that a digital micromirror device (DMD) can be exploited for fast and precise control of illumination beams for ODT. In this work, we systematically study the precision and stability of the ODT system equipped with a DMD and present measurements of 3-D and 4-D RI maps of various types of live cells including human red blood cells, white blood cells, hepatocytes, and HeLa cells. Furthermore, we also demonstrate the effective visualization of 3-D RI maps of live cells utilizing the measured information about the values and gradient of RI tomograms.

Non-invasive optoacoustic probing of the density and stiffness of single biological cells

NASA Astrophysics Data System (ADS)

Dehoux, T.; Audoin, B.

2012-12-01

Recently, the coherent generation of GHz acoustic waves using ultrashort laser pulses has demonstrated the ability to probe the sound velocity in vegetal cells and in cell-mimicking soft micro-objects with micrometer resolution, opening tremendous potentialities for single-cell biology. However, manipulating biological media in physiological conditions is often a technical challenge when using a laser-based setup. In this article, we present a new opto-acoustic bio-transducer composed of a thin metal film sputtered on a transparent heat sink that allows reducing importantly the laser-induced cellular stresses, and offers a wide variety of optical configurations. In particular, by exploiting the acoustic reflection coefficient at the sample-transducer interface and the photoacoustic interaction inside the transparent sample, the density and compressibility of the sample can be probed simultaneously. Using an ad hoc signal analysis based on Hilbert and wavelet transforms, these quantities are measured accurately for a reference fluid. Similar analysis performed in a single vegetal cell also suggests high sensitivity to the state of the transducer-cell interface, and notably to the presence of the plasma membrane that encloses the cell vacuole.

The use of FDTD in establishing in vitro experimentation conditions representative of lifelike cell phone radiation on the spermatozoa.

PubMed

Mouradi, Rand; Desai, Nisarg; Erdemir, Ahmet; Agarwal, Ashok

2012-01-01

Recent studies have shown that exposing human semen samples to cell phone radiation leads to a significant decline in sperm parameters. In daily living, a cell phone is usually kept in proximity to the groin, such as in a trouser pocket, separated from the testes by multiple layers of tissue. The aim of this study was to calculate the distance between cell phone and semen sample to set up an in vitro experiment that can mimic real life conditions (cell phone in trouser pocket separated by multiple tissue layers). For this reason, a computational model of scrotal tissues was designed by considering these separating layers, the results of which were used in a series of simulations using the Finite Difference Time Domain (FDTD) method. To provide an equivalent effect of multiple tissue layers, these results showed that the distance between a cell phone and semen sample should be 0.8 cm to 1.8 cm greater than the anticipated distance between a cell phone and the testes.

A sealable ultrathin window sample cell for the study of liquids by means of soft X-ray spectroscopy

NASA Astrophysics Data System (ADS)

Grötzsch, D.; Streeck, C.; Nietzold, C.; Malzer, W.; Mantouvalou, I.; Nutsch, A.; Dietrich, P.; Unger, W.; Beckhoff, B.; Kanngießer, B.

2017-12-01

A new sample cell concept for the analysis of liquids or solid-liquid interfaces using soft X-ray spectroscopy is presented, which enables the complete sealing of the cell as well as the transport into vacuum via, for example, a load-lock system. The cell uses pressure monitoring and active as well as passive pressure regulation systems, thereby facilitating the full control over the pressure during filling, sealing, evacuation, and measurement. The cell design and sample preparation as well as the crucial sealing procedure are explained in detail. As a first proof-of-principle experiment, successful nitrogen K-edge fluorescence yield near-edge X-ray absorption fine structure experiments of a biomolecular solution are presented. For this purpose, it is shown that the careful evaluation of all involved parameters, such as window type or photon flux, is desirable for optimizing the experimental result.

Electrospun TiO₂ nanofibers decorated Ti substrate for biomedical application.

PubMed

Dumitriu, Cristina; Stoian, Andrei Bogdan; Titorencu, Irina; Pruna, Vasile; Jinga, Victor V; Latonen, Rose-Marie; Bobacka, Johan; Demetrescu, Ioana

2014-12-01

Various TiO2 nanofibers on Ti surface have been fabricated via electrospinning and calcination. Due to different elaboration conditions the electrospun fibers have different surface feature morphologies, characterized by scanning electronic microscopy, surface roughness, and contact angle measurements. The results have indicated that the average sample diameters are between 32 and 44 nm, roughness between 61 and 416 nm, and all samples are hydrophilic. As biological evaluation, cell culture with MG63 cell line originally derived from a human osteosarcoma was performed and correlation between nanofibers elaboration, properties and cell response was established. The cell adherence and growth are more evident on Ti samples with more aligned fibers, higher roughness and strong hydrophilic character and such fibers have been elaborated with a high speed rotating cylinder collector, confirming the idea that nanostructure elaboration conditions guide the cells' growth. Copyright © 2014 Elsevier B.V. All rights reserved.

A supporting role of Chinese National Immortalized Cell Bank in life science research.

PubMed

Xu, Chong-feng; Duan, Zi-yuan

2017-01-20

A biorepository of human samples is essential to support the research of life science. Lymphoblastoid B cell line (LCL), which is easy to be prepared and can reproduce indefinitely, is a convenient form of sample preservation. LCLs are established from human B cells transformed by Epstein-Barr virus (EBV). Chinese National Immortalized Cell Bank has preserved human LCLs from different ethnic groups in China. As there are many studies on the nature of LCLs and public available resources with genome-wide data for LCLs, they have been widely applied in genetics, immunology, pharmacogenetics/genomics, regenerative medicine, cancer pathogenesis and immunotherapy, screening and generation of fully human neutralizing monoclonal antibodies and study on EBV pathogenesis. Here, we review the characteristics of LCLs and their contributions to scientific research, and introduce preserved samples in Chinese National Immortalized Cell Bank to the scientific community. We hope this bank can support more areas in the scientific research.

Hydrodynamic Assists Magnetophoreses Rare Cancer cells Separation in Microchannel Simulation and Experimental Verifications

NASA Astrophysics Data System (ADS)

Saeed, O.; Duru, L.; Yulin, D.

2018-05-01

A proposed microfluidic design has been fabricated and simulated using COMSOL Multiphysics software, based on two physical models included in this design. The device’s ability to create a narrow stream of the core sample by controlling the sheath flow rates Qs1 and Qs2 in both peripheral channels was investigated. The main target of this paper is to study the possibility of combing the hydrodynamic and magnetic techniques, in order to achieve a high rate of cancer cells separation from a cell mixture and/or buffer sample. The study has been conducted in two stages, firstly, the effects of the sheath flow rates (Qs1 and Qs2) on the sample stream focusing were studied, to find the proposed device effectiveness optimal conditions and its capability in cell focusing, and then the magnetic mechanism has been utilized to finalize the pre-labelled cells separation process.

NK sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogbomo, Henry; Hahn, Anke; Geiler, Janina

2006-01-06

The measurement of natural killer (NK) cells toxicity against tumor or virus-infected cells especially in cases with small blood samples requires highly sensitive methods. Here, a coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase release from injured target cells was used to evaluate the cytotoxicity of interleukin-2 activated NK cells against neuroblastoma cell lines. In contrast to most other methods, CLM does not require the pretreatment of target cells with labeling substances which could be toxic or radioactive. The effective killing of tumor cells was achieved by low effector/target ratios ranging from 0.5:1 to 4:1. CLM provides highly sensitive, safe,more » and fast procedure for measurement of NK cell activity with small blood samples such as those obtained from pediatric patients.« less

Expression of EphA2 protein is positively associated with age, tumor size and Fuhrman nuclear grade in clear cell renal cell carcinomas.

PubMed

Wang, Longxin; Hu, Haibing; Tian, Feng; Zhou, Wenquan; Zhou, Shuigen; Wang, Jiandong

2015-01-01

The receptor tyrosine kinase of EphA2 has been shown frequently overexpressed in various types of human carcinomas, which implicated that it plays important roles in carcinogenesis. Although EphA2 protein expression has been investigated in many types of human carcinomas, the relationship between the expression of EphA2 protein in clear cell renal cell carcinoma was not well documented. In the present study, using specific anit-EphA2 polyclonal antibody and immunohistochemistry, we evaluated EphA2 protein expression levels in clear cell RCC specimens surgically resected from 90 patients. Our results shows that EphA2 protein was positively expressed in all normal renal tubes of 90 samples (100%, 3+), which was expressed at low levels in renal cortex but high levels in the collecting ducts of the renal medulla and papilla. EphA2 was negatively or weakly expressed in 30 out of 90 samples (33.3%, 0/1+), moderately expressed in 24 samples (26.7%, 2+) and strongly expressed in 36 samples (40%, 3+). Expression of EphA2 was positively associated with age (P=0.029), tumor diameters (P<0.001) and Fuhrman nuclear grade (P<0.001). Our results indicate that EphA2 variably expressed in clear cell renal cell carcinomas. High expression of EphA2 was more often found in big size and high nuclear grade tumors, which indicated EphA2 protein may be used as a new marker for the prognosis of clear cell renal cell carcinoma.

A magnetic micropore chip for rapid (<1 hour) unbiased circulating tumor cell isolation and in situ RNA analysis.

PubMed

Ko, Jina; Bhagwat, Neha; Yee, Stephanie S; Black, Taylor; Redlinger, Colleen; Romeo, Janae; O'Hara, Mark; Raj, Arjun; Carpenter, Erica L; Stanger, Ben Z; Issadore, David

2017-09-12

The use of microtechnology for the highly selective isolation and sensitive detection of circulating tumor cells has shown enormous promise. One challenge for this technology is that the small feature sizes - which are the key to this technology's performance - can result in low sample throughput and susceptibility to clogging. Additionally, conventional molecular analysis of CTCs often requires cells to be taken off-chip for sample preparation and purification before analysis, leading to the loss of rare cells. To address these challenges, we have developed a microchip platform that combines fast, magnetic micropore based negative immunomagnetic selection (>10 mL h -1 ) with rapid on-chip in situ RNA profiling (>100× faster than conventional RNA labeling). This integrated chip can isolate both rare circulating cells and cell clusters directly from whole blood and allow individual cells to be profiled for multiple RNA cancer biomarkers, achieving sample-to-answer in less than 1 hour for 10 mL of whole blood. To demonstrate the power of this approach, we applied our device to the circulating tumor cell based diagnosis of pancreatic cancer. We used a genetically engineered lineage-labeled mouse model of pancreatic cancer (KPCY) to validate the performance of our chip. We show that in a cohort of patient samples (N = 25) that this device can detect and perform in situ RNA analysis on circulating tumor cells in patients with pancreatic cancer, even in those with extremely sparse CTCs (<1 CTC mL -1 of whole blood).

Sample to answer visualization pipeline for low-cost point-of-care blood cell counting

NASA Astrophysics Data System (ADS)

Smith, Suzanne; Naidoo, Thegaran; Davies, Emlyn; Fourie, Louis; Nxumalo, Zandile; Swart, Hein; Marais, Philip; Land, Kevin; Roux, Pieter

2015-03-01

We present a visualization pipeline from sample to answer for point-of-care blood cell counting applications. Effective and low-cost point-of-care medical diagnostic tests provide developing countries and rural communities with accessible healthcare solutions [1], and can be particularly beneficial for blood cell count tests, which are often the starting point in the process of diagnosing a patient [2]. The initial focus of this work is on total white and red blood cell counts, using a microfluidic cartridge [3] for sample processing. Analysis of the processed samples has been implemented by means of two main optical visualization systems developed in-house: 1) a fluidic operation analysis system using high speed video data to determine volumes, mixing efficiency and flow rates, and 2) a microscopy analysis system to investigate homogeneity and concentration of blood cells. Fluidic parameters were derived from the optical flow [4] as well as color-based segmentation of the different fluids using a hue-saturation-value (HSV) color space. Cell count estimates were obtained using automated microscopy analysis and were compared to a widely accepted manual method for cell counting using a hemocytometer [5]. The results using the first iteration microfluidic device [3] showed that the most simple - and thus low-cost - approach for microfluidic component implementation was not adequate as compared to techniques based on manual cell counting principles. An improved microfluidic design has been developed to incorporate enhanced mixing and metering components, which together with this work provides the foundation on which to successfully implement automated, rapid and low-cost blood cell counting tests.

Transmission electron microscopy artifacts in characterization of the nanomaterial-cell interactions.

PubMed

Leung, Yu Hang; Guo, Mu Yao; Ma, Angel P Y; Ng, Alan M C; Djurišić, Aleksandra B; Degger, Natalie; Leung, Frederick C C

2017-07-01

We investigated transmission electron microscopy artifacts obtained using standard sample preparation protocols applied to the investigation of Escherichia coli cells exposed to common nanomaterials, such as TiO 2 , Ag, ZnO, and MgO. While the common protocols for some nanomaterials result only in known issues of nanomaterial-independent generation of anomalous deposits due to fixation and staining, for others, there are reactions between the nanomaterial and chemicals used for post-fixation or staining. Only in the case of TiO 2 do we observe only the known issues of nanomaterial-independent generation of anomalous deposits due to exceptional chemical stability of this material. For the other three nanomaterials, different artifacts are observed. For each of those, we identify causes of the observed problems and suggest alternative sample preparation protocols to avoid artifacts arising from the sample preparation, which is essential for correct interpretation of the obtained images and drawing correct conclusions on cell-nanomaterial interactions. Finally, we propose modified sample preparation and characterization protocols for comprehensive and conclusive investigations of nanomaterial-cell interactions using electron microscopy and for obtaining clear and unambiguous revelation whether the nanomaterials studied penetrate the cells or accumulate at the cell membranes. In only the case of MgO and ZnO, the unambiguous presence of Zn and Mg could be observed inside the cells.

Study of P21 Expression in Oral Lichen Planus and Oral Squamous Cell Carcinoma by Immunohistochemical Technique.

PubMed

Baghaei, Fahimeh; Shojaei, Setareh; Afshar-Moghaddam, Noushin; Zargaran, Massoumeh; Rastin, Verisheh; Nasr, Mohsen; Moghimbeigi, Abbas

2015-09-01

Lichen planus is a mucocutaneous disease that is relatively common in middle aged individuals. Some studies have shown that oral lichen planus has a potential to progress to squamous cell carcinoma.p21 is a cyclin-dependent kinase inhibitor that regulates the cell cycle, thus it acts as an inhibitor in cell proliferation. This study was aimed to evaluate and compare the immunostaining of p21 (as a proliferation inhibitory factor) in oral lichen planus (OLP) and oral squamous cell carcinoma (OSCC). In this descriptive cross-sectional study, p21expression was investigated in 24 samples of oral lichen planus (OLP), 24 samples of oral squamous cell carcinoma (OSCC) and 24 samples of oral epithelial hyperplasia (OEH) by employing immunohistochemical staining. The mean percentage of p21-positive cells in OSCC (54.5±6.6) was significantly higher than that in OLP (32.8±6.08) and OEH (9.4±3.8). Moreover, OLP samples expressed p21 significantly higher than the OEH. Kruskal Wallis test revealed a statistically significant difference between the groups regarding the intensity of staining (p< 0.001). According to the findings of this study, the expression of p21 might be related to the potential carcinogenic transformation of lichen planus to SCC. Therefore, continuous follow-up periods for OLP are recommended for diagnosis of the malignant transformations in early stages.

Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells

PubMed Central

Lim, Eelin L.; Tomita, Aoy V.; Thilly, William G.; Polz, Martin F.

2001-01-01

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 104 cells ml−1. The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells ml−1 by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples. PMID:11525983

Controlled ice nucleation in cryopreservation--a review.

PubMed

Morris, G John; Acton, Elizabeth

2013-04-01

We review here for the first time, the literature on control of ice nucleation in cryopreservation. Water and aqueous solutions have a tendency to undercool before ice nucleation occurs. Control of ice nucleation has been recognised as a critical step in the cryopreservation of embryos and oocytes but is largely ignored for other cell types. We review the processes of ice nucleation and crystal growth in the solution around cells and tissues during cryopreservation with an emphasis on non IVF applications. The extent of undercooling that is encountered during the cooling of various cryocontainers is defined and the methods that have been employed to control the nucleation of ice are examined. The effects of controlled ice nucleation on the structure of the sample and the outcome of cryopreservation of a range of cell types and tissues are presented and the physical events which define the cellular response are discussed. Nucleation of ice is the most significant uncontrolled variable in conventional cryopreservation leading to sample to sample variation in cell recovery, viability and function and should be controlled to allow standardisation of cryopreservation protocols for cells for biobanking, cell based assays or clinical application. This intervention allows a way of increasing viability of cells and reducing variability between samples and should be included as standard operating procedures are developed. Copyright © 2012 Elsevier Inc. All rights reserved.

An Advanced Preclinical Mouse Model for Acute Myeloid Leukemia Using Patients' Cells of Various Genetic Subgroups and In Vivo Bioluminescence Imaging

PubMed Central

Vick, Binje; Rothenberg, Maja; Sandhöfer, Nadine; Carlet, Michela; Finkenzeller, Cornelia; Krupka, Christina; Grunert, Michaela; Trumpp, Andreas; Corbacioglu, Selim; Ebinger, Martin; André, Maya C.; Hiddemann, Wolfgang; Schneider, Stephanie; Subklewe, Marion; Metzeler, Klaus H.; Spiekermann, Karsten; Jeremias, Irmela

2015-01-01

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups. PMID:25793878

Malignant human cell transformation of Marcellus shale gas drilling flow back water

PubMed Central

Yao, Yixin; Chen, Tingting; Shen, Steven S.; Niu, Yingmei; DesMarais, Thomas L; Linn, Reka; Saunders, Eric; Fan, Zhihua; Lioy, Paul; Kluz, Thomas; Chen, Lung-Chi; Wu, Zhuangchun; Costa, Max

2015-01-01

The rapid development of high-volume horizontal hydraulic fracturing for mining natural gas from shale has posed potential impacts on human health and biodiversity. The produced flow back waters after hydraulic stimulation is known to carry high levels of saline and total dissolved solids. To understand the toxicity and potential carcinogenic effects of these waste waters, flow back water from five Marcellus hydraulic fracturing oil and gas wells were analyzed. The physicochemical nature of these samples was analyzed by inductively coupled plasma mass spectrometry and scanning electron microscopy / energy dispersive X-ray spectroscopy. A cytotoxicity study using colony formation as the endpoint was carried out to define the LC50 values of test samples using human bronchial epithelial cells (BEAS-2B). The BEAS-2B cell transformation assay was employed to assess the carcinogenic potential of the samples. Barium and strontium were among the most abundant metals in these samples and the same metals were found elevated in BEAS-2B cells after long-term treatment. BEAS-2B cells treated for 6 weeks with flow back waters produced colony formation in soft agar that was concentration dependant. In addition, flow back water-transformed BEAS-2B cells show a better migration capability when compared to control cells. This study provides information needed to assess the potential health impact of post-hydraulic fracturing flow back waters from Marcellus Shale natural gas mining. PMID:26210350

Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing.

PubMed

Salvianti, Francesca; Rotunno, Giada; Galardi, Francesca; De Luca, Francesca; Pestrin, Marta; Vannucchi, Alessandro Maria; Di Leo, Angelo; Pazzagli, Mario; Pinzani, Pamela

2015-09-01

The purpose of the study was to explore the feasibility of a protocol for the isolation and molecular characterization of single circulating tumor cells (CTCs) from cancer patients using a single-cell next generation sequencing (NGS) approach. To reach this goal we used as a model an artificial sample obtained by spiking a breast cancer cell line (MDA-MB-231) into the blood of a healthy donor. Tumor cells were enriched and enumerated by CellSearch(®) and subsequently isolated by DEPArray™ to obtain single or pooled pure samples to be submitted to the analysis of the mutational status of multiple genes involved in cancer. Upon whole genome amplification, samples were analysed by NGS on the Ion Torrent PGM™ system (Life Technologies) using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies), designed to investigate genomic "hot spot" regions of 50 oncogenes and tumor suppressor genes. We successfully sequenced five single cells, a pool of 5 cells and DNA from a cellular pellet of the same cell line with a mean depth of the sequencing reaction ranging from 1581 to 3479 reads. We found 27 sequence variants in 18 genes, 15 of which already reported in the COSMIC or dbSNP databases. We confirmed the presence of two somatic mutations, in the BRAF and TP53 gene, which had been already reported for this cells line, but also found new mutations and single nucleotide polymorphisms. Three variants were common to all the analysed samples, while 18 were present only in a single cell suggesting a high heterogeneity within the same cell line. This paper presents an optimized workflow for the molecular characterization of multiple genes in single cells by NGS. The described pipeline can be easily transferred to the study of single CTCs from oncologic patients.

CMV-specific T cell isolation from G-CSF mobilized peripheral blood: depletion of myeloid progenitors eliminates non-specific binding of MHC-multimers.

PubMed

Beloki, Lorea; Ciaurriz, Miriam; Mansilla, Cristina; Zabalza, Amaya; Perez-Valderrama, Estela; Samuel, Edward R; Lowdell, Mark W; Ramirez, Natalia; Olavarria, Eduardo

2014-11-19

Cytomegalovirus (CMV)-specific T cell infusion to immunocompromised patients following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is able to induce a successful anti-viral response. These cells have classically been manufactured from steady-state apheresis samples collected from the donor in an additional harvest prior to G-CSF mobilization, treatment that induces hematopoietic stem cell (HSC) mobilization to the periphery. However, two closely-timed cellular collections are not usually available in the unrelated donor setting, which limits the accessibility of anti-viral cells for adoptive immunotherapy. CMV-specific cytotoxic T cell (CTL) manufacture from the same G-CSF mobilized donor stem cell harvest offers great regulatory advantages, but the isolation using MHC-multimers is hampered by the high non-specific binding to myeloid progenitors, which reduces the purity of the cellular product. In the present study we describe an easy and fast method based on plastic adherence to remove myeloid cell subsets from 11 G-CSF mobilized donor samples. CMV-specific CTLs were isolated from the non-adherent fraction using pentamers and purity and yield of the process were compared to products obtained from unmanipulated samples. After the elimination of unwanted cell subtypes, non-specific binding of pentamers was notably reduced. Accordingly, following the isolation process the purity of the obtained cellular product was significantly improved. G-CSF mobilized leukapheresis samples can successfully be used to isolate antigen-specific T cells with MHC-multimers to be adoptively transferred following allo-HSCT, widening the accessibility of this therapy in the unrelated donor setting. The combination of the clinically translatable plastic adherence process to the antigen-specific cell isolation using MHC-multimers improves the quality of the therapeutic cellular product, thereby reducing the clinical negative effects associated with undesired alloreactive cell infusion.

Different tissue phagocytes sample apoptotic cells to direct distinct homeostasis programs

PubMed Central

Cummings, Ryan J.; Barbet, Gaetan; Bongers, Gerold; Hartmann, Boris M.; Gettler, Kyle; Muniz, Luciana; Furtado, Glaucia C.; Cho, Judy; Lira, Sergio A.; Blander, J. Magarian

2017-01-01

Recognition and removal of apoptotic cells by professional phagocytes, including dendritic cells and macrophages, preserves immune self-tolerance and prevents chronic inflammation and autoimmune pathologies1,2. The diverse array of phagocytes that reside within different tissues, combined with the necessarily prompt nature of apoptotic cell clearance, makes it difficult to study this process in situ. The full spectrum of functions executed by tissue-resident phagocytes in response to homeostatic apoptosis, therefore, remains unclear. Here we show that mouse apoptotic intestinal epithelial cells (IECs), which undergo continuous renewal to maintain optimal barrier and absorptive functions3, are not merely extruded to maintain homeostatic cell numbers4, but are also sampled by a single subset of dendritic cells and two macrophage subsets within a well-characterized network of phagocytes in the small intestinal lamina propria5,6. Characterization of the transcriptome within each subset before and after in situ sampling of apoptotic IECs revealed gene expression signatures unique to each phagocyte, including macrophage-specific lipid metabolism and amino acid catabolism, and a dendritic-cell-specific program of regulatory CD4+ T-cell activation. A common ‘suppression of inflammation’ signature was noted, although the specific genes and pathways involved varied amongst dendritic cells and macrophages, reflecting specialized functions. Apoptotic IECs were trafficked to mesenteric lymph nodes exclusively by the dendritic cell subset and served as critical determinants for the induction of tolerogenic regulatory CD4+ T-cell differentiation. Several of the genes that were differentially expressed by phagocytes bearing apoptotic IECs overlapped with susceptibility genes for inflammatory bowel disease7. Collectively, these findings provide new insights into the consequences of apoptotic cell sampling, advance our understanding of how homeostasis is maintained within the mucosa and set the stage for development of novel therapeutics to alleviate chronic inflammatory diseases such as inflammatory bowel disease. PMID:27828940

Bone marrow mesenchymal stem cell response to nano-structured oxidized and turned titanium surfaces.

PubMed

Annunziata, Marco; Oliva, Adriana; Buosciolo, Antonietta; Giordano, Michele; Guida, Agostino; Guida, Luigi

2012-06-01

The aim of this study was to analyse the topographic features of a novel nano-structured oxidized titanium implant surface and to evaluate its effect on the response of human bone marrow mesenchymal stem cells (BM-MSC) compared with a traditional turned surface. The 10 × 10 × 1 mm turned (control) and oxidized (test) titanium samples (P.H.I. s.r.l.) were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) and characterized by height, spatial and hybrid roughness parameters at different dimensional ranges of analysis. Primary cultures of BM-MSC were seeded on titanium samples and cell morphology, adhesion, proliferation and osteogenic differentiation, in terms of alkaline phosphatase activity, osteocalcin synthesis and extracellular matrix mineralization, were evaluated. At SEM and AFM analyses turned samples were grooved, whereas oxidized surfaces showed a more complex micro- and nano-scaled texture, with higher values of roughness parameters. Cell adhesion and osteogenic parameters were greater on oxidized (P<0.05 at least) vs. turned surfaces, whereas the cell proliferation rate was similar on both samples. Although both control and test samples were in the range of average roughness proper of smooth surfaces, they exhibited significantly different topographic properties in terms of height, spatial and, mostly, of hybrid parameters. This different micro- and nano-structure resulted in an enhanced adhesion and differentiation of cells plated onto the oxidized surfaces. © 2011 John Wiley & Sons A/S.

Radio-frequency powered glow discharge device and method with high voltage interface

DOEpatents

Duckworth, D.C.; Marcus, R.K.; Donohue, D.L.; Lewis, T.A.

1994-06-28

A high voltage accelerating potential, which is supplied by a high voltage direct current power supply, is applied to the electrically conducting interior wall of an RF powered glow discharge cell. The RF power supply desirably is electrically grounded, and the conductor carrying the RF power to the sample held by the probe is desirably shielded completely excepting only the conductor's terminal point of contact with the sample. The high voltage DC accelerating potential is not supplied to the sample. A high voltage capacitance is electrically connected in series between the sample on the one hand and the RF power supply and an impedance matching network on the other hand. The high voltage capacitance isolates the high DC voltage from the RF electronics, while the RF potential is passed across the high voltage capacitance to the plasma. An inductor protects at least the RF power supply, and desirably the impedance matching network as well, from a short that might occur across the high voltage capacitance. The discharge cell and the probe which holds the sample are configured and disposed to prevent the probe's components, which are maintained at ground potential, from bridging between the relatively low vacuum region in communication with the glow discharge maintained within the cell on the one hand, and the relatively high vacuum region surrounding the probe and cell on the other hand. The probe and cell also are configured and disposed to prevent the probe's components from electrically shorting the cell's components. 11 figures.

Chromosome damage and cell proliferation rates in in vitro irradiated whole blood as markers of late radiation toxicity after radiation therapy to the prostate.

PubMed

Beaton, Lindsay A; Ferrarotto, Catherine; Marro, Leonora; Samiee, Sara; Malone, Shawn; Grimes, Scott; Malone, Kyle; Wilkins, Ruth C

2013-04-01

In vitro irradiated blood samples from prostate cancer patients showing late normal tissue damage were examined for lymphocyte response by measuring chromosomal aberrations and proliferation rate. Patients were selected from a randomized trial evaluating the optimal timing of dose-escalated radiation and short-course androgen deprivation therapy. Of 438 patients, 3% experienced grade 3 late radiation proctitis and were considered to be radiosensitive. Blood samples were taken from 10 of these patients along with 20 matched samples from patients with grade 0 proctitis. The samples were irradiated at 6 Gy and, along with control samples, were analyzed for dicentric chromosomes and excess fragments per cell. Cells in first and second metaphase were also enumerated to determine the lymphocyte proliferation rate. At 6 Gy, there were statistically significant differences between the radiosensitive and control cohorts for 3 endpoints: the mean number of dicentric chromosomes per cell (3.26 ± 0.31, 2.91 ± 0.32; P=.0258), the mean number of excess fragments per cell (2.27 ± 0.23, 1.43 ± 0.37; P<.0001), and the proportion of cells in second metaphase (0.27 ± 0.10, 0.46 ± 0.09; P=.0007). These results may be a valuable indicator for identifying radiosensitive patients and for tailoring radiation therapy. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

An electromagnetic compressive force by cell exciter stimulates chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

PubMed

Park, Sang-Hyug; Sim, Woo Young; Park, Sin Wook; Yang, Sang Sik; Choi, Byung Hyune; Park, So Ra; Park, Kwideok; Min, Byoung-Hyun

2006-11-01

In this study, we present a biological micro-electromechanical system and its application to the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (MSCs). Actuated by an electromagnetic force, the micro cell exciter was designed to deliver a cyclic compressive load (CCL) with various magnitudes. Two major parts in the system are an actuator and a cartridge-type chamber. The former has a permanent magnet and coil, and the latter is equipped with 7 sample dishes and 7 metal caps. Mixed with a 2.4% alginate solution, the alginate/MSC layers were positioned in the sample dishes; the caps contained chondrogenic defined medium without transforming growth factor-beta (TGF-beta). Once powered, the actuator coil-derived electromagnetic force pulled the metal caps down, compressing the samples. The cyclic load was given at 1-Hz frequency for 10 min twice a day. Samples in the dishes without a cap served as a control. The samples were analyzed at 3, 5, and 7 days after stimulation for cell viability, biochemical assays, histologic features, immunohistochemistry, and gene expression of the chondrogenic markers. Applied to the alginate/MSC layer, the CCL system enhanced the synthesis of cartilage-specific matrix proteins and the chondrogenic markers, such as aggrecan, type II collagen, and Sox9. We found that the micromechanically exerted CCL by the cell exciter was very effective in enhancing the chondrogenic differentiation of MSCs, even without using exogenous TGF-beta.

High anaplastic lymphoma kinase immunohistochemical staining in neuroblastoma and ganglioneuroblastoma is an independent predictor of poor outcome.

PubMed

Duijkers, Floor A M; Gaal, José; Meijerink, Jules P P; Admiraal, Pieter; Pieters, Rob; de Krijger, Ronald R; van Noesel, Max M

2012-03-01

Anaplastic lymphoma kinase (ALK) mutations occur in 3% to 11% of neuroblastoma (NBL) cases and are associated with high ALK levels. However, high ALK levels appear to be a mutation-independent hallmark of NBL. Evidence about the prognostic relevance of ALK mutations and ALK tumor positivity in patients with NBL has been inconclusive. In this study, we investigated the prognostic relevance of ALK positivity by IHC and ALK mutation status by PCR sequencing in 71 NBL, 12 ganglioneuroblastoma (GNBL), and 20 ganglioneuroma samples in a multivariate model. ALK mutations were present in 2 of 72 NBL and 2 of 12 GNBL samples, which all contained many ALK-positive cells (>50%). In addition, half of all NBL samples showed ALK positivity in most (>50%) of tumor cells, whereas half of the GNBL showed staining in <20% of the tumor cells. In most ganglioneuroma samples, a low percentage of tumor cells stained positive for ALK, which mainly involved ganglion cells. Higher percentages of ALK-positive cells in NBL and GNBL patient samples correlated with inferior survival in univariate and multivariate analyses with established prognostic factors, such as stage, age, and MYCN status. In conclusion, ALK positivity by IHC is an independent, poor prognostic factor in patients with GNBL and NBL. ALK IHC is an easy test suitable for future risk stratification in patients with NBL and GNBL. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Radio-frequency powered glow discharge device and method with high voltage interface

DOEpatents

Duckworth, Douglas C.; Marcus, R. Kenneth; Donohue, David L.; Lewis, Trousdale A.

1994-01-01

A high voltage accelerating potential, which is supplied by a high voltage direct current power supply, is applied to the electrically conducting interior wall of an RF powered glow discharge cell. The RF power supply desirably is electrically grounded, and the conductor carrying the RF power to the sample held by the probe is desirably shielded completely excepting only the conductor's terminal point of contact with the sample. The high voltage DC accelerating potential is not supplied to the sample. A high voltage capacitance is electrically connected in series between the sample on the one hand and the RF power supply and an impedance matching network on the other hand. The high voltage capacitance isolates the high DC voltage from the RF electronics, while the RF potential is passed across the high voltage capacitance to the plasma. An inductor protects at least the RF power supply, and desirably the impedance matching network as well, from a short that might occur across the high voltage capacitance. The discharge cell and the probe which holds the sample are configured and disposed to prevent the probe's components, which are maintained at ground potential, from bridging between the relatively low vacuum region in communication with the glow discharge maintained within the cell on the one hand, and the relatively high vacuum region surrounding the probe and cell on the other hand. The probe and cell also are configured and disposed to prevent the probe's components from electrically shorting the cell's components.

Microbiota of Cow’s Milk; Distinguishing Healthy, Sub-Clinically and Clinically Diseased Quarters

PubMed Central

Oikonomou, Georgios; Bicalho, Marcela Lucas; Meira, Enoch; Rossi, Rodolfo Elke; Foditsch, Carla; Machado, Vinicius Silva; Teixeira, Andre Gustavo Vieira; Santisteban, Carlos; Schukken, Ynte Hein; Bicalho, Rodrigo Carvalho

2014-01-01

The objective of this study was to use pyrosequencing of the 16S rRNA genes to describe the microbial diversity of bovine milk samples derived from clinically unaffected quarters across a range of somatic cell counts (SCC) values or from clinical mastitis, culture negative quarters. The obtained microbiota profiles were used to distinguish healthy, subclinically and clinically affected quarters. Two dairy farms were used for the collection of milk samples. A total of 177 samples were used. Fifty samples derived from healthy, culture negative quarters with a SCC of less than 20,000 cells/ml (group 1); 34 samples derived from healthy, culture negative quarters, with a SCC ranging from 21,000 to 50,000 cells/ml (group 2); 26 samples derived from healthy, culture negative quarters with a SCC greater than 50,000 cells/ml (group 3); 34 samples derived from healthy, culture positive quarters, with a SCC greater than 400,000 (group 4, subclinical); and 33 samples derived from clinical mastitis, culture negative quarters (group 5, clinical). Bacterial DNA was isolated from these samples and the 16S rRNA genes were individually amplified and pyrosequenced. All samples analyzed revealed great microbial diversity. Four bacterial genera were present in every sample obtained from healthy quarters (Faecalibacterium spp., unclassified Lachnospiraceae, Propionibacterium spp. and Aeribacillus spp.). Discriminant analysis models showed that samples derived from healthy quarters were easily discriminated based on their microbiota profiles from samples derived from clinical mastitis, culture negative quarters; that was also the case for samples obtained from different farms. Staphylococcus spp. and Streptococcus spp. were among the most prevalent genera in all groups while a general multivariable linear model revealed that Sphingobacterium and Streptococcus prevalences were associated with increased 10 log SCC. Conversely, Nocardiodes and Paenibacillus were negatively correlated, and a higher percentage of the genera was associated with a lower 10 log SCC. PMID:24465777

A perpetual source of DNA or something really different: ethical issues in the creation of cell lines for African genomics research.

PubMed

de Vries, Jantina; Abayomi, Akin; Brandful, James; Littler, Katherine; Madden, Ebony; Marshall, Patricia; Ouwe Missi Oukem-Boyer, Odile; Seeley, Janet

2014-08-07

The rise of genomic studies in Africa - not least due to projects funded under H3Africa - is associated with the development of a small number of biorepositories across Africa. For the ultimate success of these biorepositories, the creation of cell lines including those from selected H3Africa samples would be beneficial. In this paper, we map ethical challenges in the creation of cell lines. The first challenge we identified relates to the moral status of cells living in culture. There is no doubt that cells in culture are alive, and the question is how this characteristic is relevant to ethical decision-making. The second challenge relates to the fact that cells in culture are a source of cell products and mitochondrial DNA. In combination with other technologies, cells in culture could also be used to grow human tissue. Whilst on the one hand, this feature increases the potential utility of the sample and promotes science, on the other it also enables further scientific work that may not have been specifically consented to or approved. The third challenge relates to ownership over samples, particularly in cases where cell lines are created by a biobank, and in a different country than where samples were collected. Relevant questions here concern the export of samples, approval of secondary use and the acceptability of commercialisation. A fourth challenge relates to perceptions of blood and bodily integrity, which may be particularly relevant for African research participants from certain cultures or backgrounds. Finally, we discuss challenges around informed consent and ethical review. In this paper, we sought to map the myriad of ethical challenges that need to be considered prior to making cell line creation a reality in the H3Africa project. Considering the relative novelty of this practice in Africa, such challenges will need to be considered, discussed and potentially be resolved before cell line creation in Africa becomes financially feasible and sustainable. We suggest that discussions need to be undertaken between stakeholders internationally, considering the international character of the H3Africa project. We also map out avenues for empirical research.

Evaluation of epigenetic inactivation of vascular endothelial growth factor receptors in head and neck squamous cell carcinoma.

PubMed

Misawa, Yuki; Misawa, Kiyoshi; Kawasaki, Hideya; Imai, Atsushi; Mochizuki, Daiki; Ishikawa, Ryuji; Endo, Shiori; Mima, Masato; Kanazawa, Takeharu; Iwashita, Toshihide; Mineta, Hiroyuki

2017-07-01

The aim of this study was to determine the methylation status of the genes encoding the vascular endothelial growth factor receptors and to evaluate the usefulness of VEGFR methylation as a prognostic indicator in head and neck squamous cell carcinoma. VEGFR messenger RNA expression and promoter methylation were examined in a panel of cell lines via quantitative reverse transcription and methylation-specific polymerase chain reaction, respectively. Promoter methylation was compared with clinical characteristics in 128 head and neck squamous cell carcinoma samples. The normalized methylation values for the VEGFR1, VEGFR2 and VEGFR3 promoters tended to be higher in the tumour cell lines than in normal tonsil samples, whereas amounts of VEGFR1, VEGFR2 and VEGFR3 messenger RNA were significantly higher. Methylation of the VEGFR1 promoter (p = 0.003; 66/128 head and neck squamous cell carcinoma samples, 52%) and VEGFR3 promoter (p = 0.043; 53/128 head and neck squamous cell carcinoma samples, 41%) significantly correlated with recurrence, whereas methylation of the VEGFR2 promoter significantly correlated with lymph node metastasis (p = 0.046; 47/128 head and neck squamous cell carcinoma samples, 37%). Concurrent methylation of the VEGFR1 and VEGFR3 promoters significantly correlated with reduced disease-free survival (log-rank test, p = 0.009). In a multivariate logistic regression analysis, methylation of the VEGFR1, VEGFR3 and both the VEGFR1 and VEGFR3 promoters independently predicted recurrence (odds ratios and 95% confidence intervals: 3.19, 1.51-6.75 (p = 0.002); 2.24, 1.06-4.76 (p = 0.035); and 2.56, 1.09-6.05 (p = 0.032), respectively). Methylation of the VEGFR promoters predicts poor prognosis in head and neck squamous cell carcinoma patients.

Effect of Female Genital Schistosomiasis and Anti-Schistosomal Treatment on Monocytes, CD4+ T-Cells and CCR5 Expression in the Female Genital Tract

PubMed Central

Kleppa, Elisabeth; Ramsuran, Veron; Zulu, Siphosenkosi; Karlsen, Gunn Hege; Bere, Alfred; Passmore, Jo-Ann S.; Ndhlovu, Patricia; Lillebø, Kristine; Holmen, Sigve D.; Onsrud, Mathias; Gundersen, Svein Gunnar; Taylor, Myra; Kjetland, Eyrun F.; Ndung’u, Thumbi

2014-01-01

Background Schistosoma haematobium is a waterborne parasite that may cause female genital schistosomiasis (FGS), characterized by genital mucosal lesions. There is clinical and epidemiological evidence for a relationship between FGS and HIV. We investigated the impact of FGS on HIV target cell density and expression of the HIV co-receptor CCR5 in blood and cervical cytobrush samples. Furthermore we evaluated the effect of anti-schistosomal treatment on these cell populations. Design The study followed a case-control design with post treatment follow-up, nested in an on-going field study on FGS. Methods Blood and cervical cytobrush samples were collected from FGS negative and positive women for flow cytometry analyses. Urine samples were investigated for schistosome ova by microscopy and polymerase chain reaction (PCR). Results FGS was associated with a higher frequency of CD14+ cells (monocytes) in blood (11.5% in FGS+ vs. 2.2% in FGS-, p = 0.042). Frequencies of CD4+ cells expressing CCR5 were higher in blood samples from FGS+ than from FGS- women (4.7% vs. 1.5%, p = 0.018). The CD14+ cell population decreased significantly in both compartments after anti-schistosomal treatment (p = 0.043). Although the frequency of CD4+ cells did not change after treatment, frequencies of CCR5 expression by CD4+ cells decreased significantly in both compartments (from 3.4% to 0.5% in blood, p = 0.036; and from 42.4% to 5.6% in genital samples, p = 0.025). Conclusions The results support the hypothesis that FGS may increase the risk of HIV acquisition, not only through damage of the mucosal epithelial barrier, but also by affecting HIV target cell populations, and that anti-schistosomal treatment can modify this. PMID:24896815

Halo-free Phase Contrast Microscopy

NASA Astrophysics Data System (ADS)

Nguyen, Tan H.; Kandel, Mikhail; Shakir, Haadi M.; Best-Popescu, Catherine; Arikkath, Jyothi; Do, Minh N.; Popescu, Gabriel

2017-03-01

We present a new approach for retrieving halo-free phase contrast microscopy (hfPC) images by upgrading the conventional PC microscope with an external interferometric module, which generates sufficient data for reversing the halo artifact. Acquiring four independent intensity images, our approach first measures haloed phase maps of the sample. We solve for the halo-free sample transmission function by using a physical model of the image formation under partial spatial coherence. Using this halo-free sample transmission, we can numerically generate artifact-free PC images. Furthermore, this transmission can be further used to obtain quantitative information about the sample, e.g., the thickness with known refractive indices, dry mass of live cells during their cycles. We tested our hfPC method on various control samples, e.g., beads, pillars and validated its potential for biological investigation by imaging live HeLa cells, red blood cells, and neurons.

A New Method for Calculating Counts in Cells

NASA Astrophysics Data System (ADS)

Szapudi, István

1998-04-01

In the near future, a new generation of CCD-based galaxy surveys will enable high-precision determination of the N-point correlation functions. The resulting information will help to resolve the ambiguities associated with two-point correlation functions, thus constraining theories of structure formation, biasing, and Gaussianity of initial conditions independently of the value of Ω. As one of the most successful methods of extracting the amplitude of higher order correlations is based on measuring the distribution of counts in cells, this work presents an advanced way of measuring it with unprecedented accuracy. Szapudi & Colombi identified the main sources of theoretical errors in extracting counts in cells from galaxy catalogs. One of these sources, termed as measurement error, stems from the fact that conventional methods use a finite number of sampling cells to estimate counts in cells. This effect can be circumvented by using an infinite number of cells. This paper presents an algorithm, which in practice achieves this goal; that is, it is equivalent to throwing an infinite number of sampling cells in finite time. The errors associated with sampling cells are completely eliminated by this procedure, which will be essential for the accurate analysis of future surveys.

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy

DOE PAGES

Ducic, Tanja; Paunesku, Tatjana; Chen, Si; ...

2016-12-09

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different samplemore » preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. In conclusion, each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.« less

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ducic, Tanja; Paunesku, Tatjana; Chen, Si

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different samplemore » preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. In conclusion, each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.« less

Detection of circulating tumor cells from cryopreserved human sarcoma peripheral blood mononuclear cells.

PubMed

Li, Heming; Meng, Qing H; Noh, Hyangsoon; Batth, Izhar Singh; Somaiah, Neeta; Torres, Keila E; Xia, Xueqing; Wang, Ruoyu; Li, Shulin

2017-09-10

Circulating tumor cells (CTCs) enter the vasculature or lymphatic system after shedding from the primary tumor. CTCs may serve as "seed" cells for tumor metastasis. The utility of CTCs in clinical applications for sarcoma is not fully investigated, partly owing to the necessity for fresh blood samples and the lack of a CTC-specific antibody. To overcome these drawbacks, we developed a technique for sarcoma CTCs capture and detection using cryopreserved peripheral blood mononuclear cells (PBMCs) and our proprietary cell-surface vimentin (CSV) antibody 84-1, which is specific to tumor cells. This technique was validated by sarcoma cell spiking assay, matched CTCs comparison between fresh and cryopreserved PBMCs, and independent tumor markers in multiple types of sarcoma patient blood samples. The reproducibility was maximized when cryopreserved PBMCs were prepared from fresh blood samples within 2 h of the blood draw. In summary, as far as we are aware, ours is the first report to capture and detect CTCs from cryopreserved PBMCs. Further validation in other types of tumor may help boost the feasibility and utility of CTC-based diagnosis in a centralized laboratory. Copyright © 2017 Elsevier B.V. All rights reserved.

Sickle cell detection using a smartphone

PubMed Central

Knowlton, S. M.; Sencan, I.; Aytar, Y.; Khoory, J.; Heeney, M. M.; Ghiran, I. C.; Tasoglu, S.

2015-01-01

Sickle cell disease affects 25% of people living in Central and West Africa and, if left undiagnosed, can cause life threatening “silent” strokes and lifelong damage. However, ubiquitous testing procedures have yet to be implemented in these areas, necessitating a simple, rapid, and accurate testing platform to diagnose sickle cell disease. Here, we present a label-free, sensitive, and specific testing platform using only a small blood sample (<1 μl) based on the higher density of sickle red blood cells under deoxygenated conditions. Testing is performed with a lightweight and compact 3D-printed attachment installed on a commercial smartphone. This attachment includes an LED to illuminate the sample, an optical lens to magnify the image, and two permanent magnets for magnetic levitation of red blood cells. The sample is suspended in a paramagnetic medium with sodium metabisulfite and loaded in a microcapillary tube that is inserted between the magnets. Red blood cells are levitated in the magnetic field based on equilibrium between the magnetic and buoyancy forces acting on the cells. Using this approach, we were able to distinguish between the levitation patterns of sickle versus control red blood cells based on their degree of confinement. PMID:26492382

Sickle cell detection using a smartphone.

PubMed

Knowlton, S M; Sencan, I; Aytar, Y; Khoory, J; Heeney, M M; Ghiran, I C; Tasoglu, S

2015-10-22

Sickle cell disease affects 25% of people living in Central and West Africa and, if left undiagnosed, can cause life threatening "silent" strokes and lifelong damage. However, ubiquitous testing procedures have yet to be implemented in these areas, necessitating a simple, rapid, and accurate testing platform to diagnose sickle cell disease. Here, we present a label-free, sensitive, and specific testing platform using only a small blood sample (<1 μl) based on the higher density of sickle red blood cells under deoxygenated conditions. Testing is performed with a lightweight and compact 3D-printed attachment installed on a commercial smartphone. This attachment includes an LED to illuminate the sample, an optical lens to magnify the image, and two permanent magnets for magnetic levitation of red blood cells. The sample is suspended in a paramagnetic medium with sodium metabisulfite and loaded in a microcapillary tube that is inserted between the magnets. Red blood cells are levitated in the magnetic field based on equilibrium between the magnetic and buoyancy forces acting on the cells. Using this approach, we were able to distinguish between the levitation patterns of sickle versus control red blood cells based on their degree of confinement.

DRUG-NEM: Optimizing drug combinations using single-cell perturbation response to account for intratumoral heterogeneity

PubMed Central

Anchang, Benedict; Davis, Kara L.; Fienberg, Harris G.; Bendall, Sean C.; Karacosta, Loukia G.; Tibshirani, Robert; Nolan, Garry P.; Plevritis, Sylvia K.

2018-01-01

An individual malignant tumor is composed of a heterogeneous collection of single cells with distinct molecular and phenotypic features, a phenomenon termed intratumoral heterogeneity. Intratumoral heterogeneity poses challenges for cancer treatment, motivating the need for combination therapies. Single-cell technologies are now available to guide effective drug combinations by accounting for intratumoral heterogeneity through the analysis of the signaling perturbations of an individual tumor sample screened by a drug panel. In particular, Mass Cytometry Time-of-Flight (CyTOF) is a high-throughput single-cell technology that enables the simultaneous measurements of multiple (>40) intracellular and surface markers at the level of single cells for hundreds of thousands of cells in a sample. We developed a computational framework, entitled Drug Nested Effects Models (DRUG-NEM), to analyze CyTOF single-drug perturbation data for the purpose of individualizing drug combinations. DRUG-NEM optimizes drug combinations by choosing the minimum number of drugs that produce the maximal desired intracellular effects based on nested effects modeling. We demonstrate the performance of DRUG-NEM using single-cell drug perturbation data from tumor cell lines and primary leukemia samples. PMID:29654148

Microsystems for the Capture of Low-Abundance Cells

NASA Astrophysics Data System (ADS)

Dharmasiri, Udara; Witek, Małgorzata A.; Adams, Andre A.; Soper, Steven A.

2010-07-01

Efficient selection and enumeration of low-abundance biological cells are highly important in a variety of applications. For example, the clinical utility of circulating tumor cells (CTCs) in peripheral blood is recognized as a viable biomarker for the management of various cancers, in which the clinically relevant number of CTCs per 7.5 ml of blood is two to five. Although there are several methods for isolating rare cells from a variety of heterogeneous samples, such as immunomagnetic-assisted cell sorting and fluorescence-activated cell sorting, they are fraught with challenges. Microsystem-based technologies are providing new opportunities for selecting and isolating rare cells from complex, heterogeneous samples. Such approaches involve reductions in target-cell loss, process automation, and minimization of contamination issues. In this review, we introduce different application areas requiring rare cell analysis, conventional techniques for their selection, and finally microsystem approaches for low-abundance-cell isolation and enumeration.

Paracoccidioides brasiliensis interacts with dermal dendritic cells and keratinocytes in human skin and oral mucosa lesions.

PubMed

Silva, Wellington Luiz Ferreira da; Pagliari, Carla; Duarte, Maria Irma Seixas; Sotto, Mirian N

2016-05-01

Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. In PCM the skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to immunohistochemical and/or immunofluorescence staining with anti-factor XIIIa (marker of dermal dendrocytes), anti-CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti-P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed for better visualization of the interaction between keratinocytes and the fungi. In sum, 42% of oral mucosa samples displayed yeast forms in Factor XIIIa dermal dendrocytes cytoplasm. Langerhans cells in skin and oral mucosa samples did not show yeast cells in their cytoplasm. In sum, 54% of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms. Factor XIIIa dendrocytes and keratinocytes may be acting as antigen-presenting cells to fulfill the probably impaired function of Langerhans cells in skin and oral mucosa of human PCM. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Laser capture microdissection of embryonic cells and preparation of RNA for microarray assays.

PubMed

Redmond, Latasha C; Pang, Christopher J; Dumur, Catherine; Haar, Jack L; Lloyd, Joyce A

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice-isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure(®) LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.

Radiosensitization by inhibiting STAT1 in renal cell carcinoma.

PubMed

Hui, Zhouguang; Tretiakova, Maria; Zhang, Zhongfa; Li, Yan; Wang, Xiaozhen; Zhu, Julie Xiaohong; Gao, Yuanhong; Mai, Weiyuan; Furge, Kyle; Qian, Chao-Nan; Amato, Robert; Butler, E Brian; Teh, Bin Tean; Teh, Bin S

2009-01-01

Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy, but the molecular mechanism underlying its radioresistance is not understood. This study investigated the role of signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, in radioresistant RCC. The expressions of STAT1 and STAT3 in 164 human clear cell RCC samples, 47 papillary RCC samples, and 15 normal kidney tissue samples were examined by microarray expression profiling and immunohistochemistry. Western blotting was performed to evaluate the total and phosphorylated STAT1 expression in CRL-1932 (786-O) (human clear cell RCC), SKRC-39 (human papillary RCC), CCL-116 (human fibroblast), and CRL-1441 (G-401) (human Wilms tumor). STAT1 was reduced or inhibited by fludarabine and siRNA, respectively, and the effects on radiation-induced cell death were investigated using clonogenic assays. STAT1 expression, but not STAT3 expression, was significantly greater in human RCC samples (p = 1.5 x 10(-8) for clear cell; and p = 3.6 x 10(-4) for papillary). Similarly, the expression of STAT1 was relatively greater in the two RCC cell lines. STAT1 expression was reduced by both fludarabine and siRNA, significantly increasing the radiosensitivity in both RCC cell lines. This is the first study reporting the overexpression of STAT1 in human clear cell and papillary RCC tissues. Radiosensitization in RCC cell lines was observed by a reduction or inhibition of STAT1 signaling, using fludarabine or siRNA. Our data suggest that STAT1 may play a key role in RCC radioresistance and manipulation of this pathway may enhance the efficacy of radiotherapy.

Laser Capture Microdissection of Embryonic Cells and Preparation of RNA for Microarray Assays

PubMed Central

Redmond, Latasha C.; Pang, Christopher J.; Dumur, Catherine; Haar, Jack L.; Lloyd, Joyce A.

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice–isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure® LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM. PMID:24318813

Rapid detection of Naegleria fowleri in water distribution pipeline biofilms and drinking water samples.

PubMed

Puzon, Geoffrey J; Lancaster, James A; Wylie, Jason T; Plumb, Iason J

2009-09-01

Rapid detection of pathogenic Naegleria fowler in water distribution networks is critical for water utilities. Current detection methods rely on sampling drinking water followed by culturing and molecular identification of purified strains. This culture-based method takes an extended amount of time (days), detects both nonpathogenic and pathogenic species, and does not account for N. fowleri cells associated with pipe wall biofilms. In this study, a total DNA extraction technique coupled with a real-time PCR method using primers specific for N. fowleri was developed and validated. The method readily detected N. fowleri without preculturing with the lowest detection limit for N. fowleri cells spiked in biofilm being one cell (66% detection rate) and five cells (100% detection rate). For drinking water, the detection limit was five cells (66% detection rate) and 10 cells (100% detection rate). By comparison, culture-based methods were less sensitive for detection of cells spiked into both biofilm (66% detection for <10 cells) and drinking water (0% detection for <10 cells). In mixed cultures of N. fowleri and nonpathogenic Naegleria, the method identified N. fowleri in 100% of all replicates, whereastests with the current consensus primers detected N. fowleri in only 5% of all replicates. Application of the new method to drinking water and pipe wall biofilm samples obtained from a distribution network enabled the detection of N. fowleri in under 6 h, versus 3+ daysforthe culture based method. Further, comparison of the real-time PCR data from the field samples and the standard curves enabled an approximation of N. fowleri cells in the biofilm and drinking water. The use of such a method will further aid water utilities in detecting and managing the persistence of N. fowleri in water distribution networks.

COX-2 expression in canine anal sac adenocarcinomas and in non-neoplastic canine anal sacs.

PubMed

Knudsen, C S; Williams, A; Brearley, M J; Demetriou, J L

2013-09-01

Anal sac adenocarcinoma (ASAC) is a clinically significant canine neoplasm characterized by early lymphatic invasion. Up-regulation of cyclooxygenase isoform 2 (COX-2) has been confirmed in several animal and human neoplastic tissues. The aim of the current study was primarily to evaluate COX-2 expression in canine ASAC and compare it to COX-2 expression in non-neoplastic canine anal sac tissue using immunohistochemistry with scoring for percentage positivity and intensity. Twenty-five ASAC samples and 22 normal anal sacs were available for evaluation. All canine ASAC samples and the normal anal sac tissues stained positively for COX-2. However, while normal anal sac tissue showed strong staining of the ductal epithelial cells, ASAC samples showed staining of the neoplastic glandular epithelial cells, with varying percentage positivity and intensity between ASAC samples. COX-2 immunoreactivity of ASAC samples was of low intensity in 52% and high in 12% of the cases; the remaining samples were of intermediate intensity. Seventy-six per cent of the ASAC had over 50% of the neoplastic glandular cells staining positive. These results confirm that COX-2 is expressed in the neoplastic glandular epithelial cells in canine ASAC and suggest a potential role for COX-2 inhibitors in the management of ASAC. Furthermore, the results indicate that COX-2 is expressed in ductal epithelial cells of the normal anal sac. Copyright © 2013 Elsevier Ltd. All rights reserved.

Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Ying; Zhao, Rui; Piehowski, Paul D.

One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-µm-i.d. columns increase signal intensity by >3-fold relative to those using 75-µm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos mass spectrometer significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading tomore » a ~3× increase in peptide identifications and 1.7× increase in identified protein groups for 2 ng tryptic digests of bacterial lysate. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~ 95% for 0.5 ng samples and by ~42% for 2 ng samples. The present platform is capable of identifying >3000 protein groups from tryptic digestion of cell lysates equivalent to 50 HeLa cells and 100 THP-1 cells (~10 ng total proteins), respectively, and >950 proteins from subnanogram bacterial and archaeal cell lysates. The present ultrasensitive LC-MS platform is expected to enable deep proteome coverage for subnanogram samples, including single mammalian cells.« less

A new and standardized method to sample and analyse vitreous samples by the Cellient automated cell block system.

PubMed

Van Ginderdeuren, Rita; Van Calster, Joachim; Stalmans, Peter; Van den Oord, Joost

2014-08-01

In this prospective study, a universal protocol for sampling and analysing vitreous material was investigated. Vitreous biopsies are difficult to handle because of the paucity of cells and the gelatinous structure of the vitreous. Histopathological analysis of the vitreous is useful in difficult uveitis cases to differentiate uveitis from lymphoma or infection and to define the type of cellular reaction. Hundred consecutive vitreous samples were analysed with the Cellient tissue processor (Hologic). This machine is a fully automated processor starting from a specified container with PreservCyt (fixative fluid) with cells to paraffin. Cytology was compared with fixatives Cytolyt (contains a mucolyticum) and PreservCyt. Routine histochemical and immunostainings were evaluated. In 92% of the cases, sufficient material was found for diagnosis. In 14%, a Cytolyt wash was necessary to prevent clotting of the tubes in the Cellient due to the viscosity of the sample. In 23%, the diagnosis was an acute inflammation (presence of granulocytes); in 33%, chronic active inflammation (presence of T lymphocytes); in 33%, low-grade inflammation (presence of CD68 cells, without T lymphocytes); and in 3%, a malignant process. A standardized protocol for sampling and handling vitreous biopsies, fixing in PreservCyt and processing by the Cellient gives a satisfactory result in morphology, number of cells and possibility of immuno-histochemical stainings. The diagnosis can be established or confirmed in more than 90% of cases. © 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Real-time detection of BRAF V600E mutation from archival hairy cell leukemia FFPE tissue by nanopore sequencing.

PubMed

Vacca, Davide; Cancila, Valeria; Gulino, Alessandro; Lo Bosco, Giosuè; Belmonte, Beatrice; Di Napoli, Arianna; Florena, Ada Maria; Tripodo, Claudio; Arancio, Walter

2018-02-01

The MinION is a miniaturized high-throughput next generation sequencing platform of novel conception. The use of nucleic acids derived from formalin-fixed paraffin-embedded samples is highly desirable, but their adoption for molecular assays is hurdled by the high degree of fragmentation and by the chemical-induced mutations stemming from the fixation protocols. In order to investigate the suitability of MinION sequencing on formalin-fixed paraffin-embedded samples, the presence and frequency of BRAF c.1799T > A mutation was investigated in two archival tissue specimens of Hairy cell leukemia and Hairy cell leukemia Variant. Despite the poor quality of the starting DNA, BRAF mutation was successfully detected in the Hairy cell leukemia sample with around 50% of the reads obtained within 2 h of the sequencing start. Notably, the mutational burden of the Hairy cell leukemia sample as derived from nanopore sequencing proved to be comparable to a sensitive method for the detection of point mutations, namely the Digital PCR, using a validated assay. Nanopore sequencing can be adopted for targeted sequencing of genetic lesions on critical DNA samples such as those extracted from archival routine formalin-fixed paraffin-embedded samples. This result let speculating about the possibility that the nanopore sequencing could be trustably adopted for the real-time targeted sequencing of genetic lesions. Our report opens the window for the adoption of nanopore sequencing in molecular pathology for research and diagnostics.

Droplet microfluidics for amplification-free genetic detection of single cells.

PubMed

Rane, Tushar D; Zec, Helena C; Puleo, Chris; Lee, Abraham P; Wang, Tza-Huei

2012-09-21

In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.

Back surface reflectors for solar cells

NASA Technical Reports Server (NTRS)

Chai, A. T.

1980-01-01

Sample solar cells were fabricated to study the effects of various back surface reflectors on the device performance. They are typical 50 micrometers thick, space quality, silicon solar cells except for variations of the back contact configuration. The back surfaces of the sample cells are polished to a mirror like finish, and have either conventional full contacts or grid finger contacts. Measurements and evaluation of various metallic back surface reflectors, as well as cells with total internal reflection, are presented. Results indicate that back surface reflectors formed using a grid finger back contact are more effective reflectors than cells with full back metallization and that Au, Ag, or Cu are better back surface reflector metals than Al.

Leukaemia cell of origin identified by chromatin landscape of bulk tumour cells

PubMed Central

George, Joshy; Uyar, Asli; Young, Kira; Kuffler, Lauren; Waldron-Francis, Kaiden; Marquez, Eladio; Ucar, Duygu; Trowbridge, Jennifer J.

2016-01-01

The precise identity of a tumour's cell of origin can influence disease prognosis and outcome. Methods to reliably define tumour cell of origin from primary, bulk tumour cell samples has been a challenge. Here we use a well-defined model of MLL-rearranged acute myeloid leukaemia (AML) to demonstrate that transforming haematopoietic stem cells (HSCs) and multipotent progenitors results in more aggressive AML than transforming committed progenitor cells. Transcriptome profiling reveals a gene expression signature broadly distinguishing stem cell-derived versus progenitor cell-derived AML, including genes involved in immune escape, extravasation and small GTPase signal transduction. However, whole-genome profiling of open chromatin reveals precise and robust biomarkers reflecting each cell of origin tested, from bulk AML tumour cell sampling. We find that bulk AML tumour cells exhibit distinct open chromatin loci that reflect the transformed cell of origin and suggest that open chromatin patterns may be leveraged as prognostic signatures in human AML. PMID:27397025

Patient-derived Mammosphere and Xenograft Tumour Initiation Correlates with Progression to Metastasis.

PubMed

Eyre, Rachel; Alférez, Denis G; Spence, Kath; Kamal, Mohamed; Shaw, Frances L; Simões, Bruno M; Santiago-Gómez, Angélica; Sarmiento-Castro, Aida; Bramley, Maria; Absar, Mohammed; Saad, Zahida; Chatterjee, Sumohan; Kirwan, Cliona; Gandhi, Ashu; Armstrong, Anne C; Wardley, Andrew M; O'Brien, Ciara S; Farnie, Gillian; Howell, Sacha J; Clarke, Robert B

2016-12-01

Breast cancer specific mortality results from tumour cell dissemination and metastatic colonisation. Identification of the cells and processes responsible for metastasis will enable better prevention and control of metastatic disease, thus reducing relapse and mortality. To better understand these processes, we prospectively collected 307 patient-derived breast cancer samples (n = 195 early breast cancers (EBC) and n = 112 metastatic samples (MBC)). We assessed colony-forming activity in vitro by growing isolated cells in both primary (formation) and secondary (self-renewal) mammosphere culture, and tumour initiating activity in vivo through subcutaneous transplantation of fragments or cells into mice. Metastatic samples formed primary mammosphere colonies significantly more frequently than early breast cancers and had significantly higher primary mammosphere colony formation efficiency (0.9 % vs. 0.6 %; p < 0.0001). Tumour initiation in vivo was significantly higher in metastatic than early breast cancer samples (63 % vs. 38 %, p = 0.04). Of 144 breast cancer samples implanted in vivo, we established 20 stable patient-derived xenograft (PDX) models at passage 2 or greater. Lung metastases were detected in mice from 14 PDX models. Mammosphere colony formation in vitro significantly correlated with the ability of a tumour to metastasise to the lungs in vivo (p = 0.05), but not with subcutaneous tumour initiation. In summary, the breast cancer stem cell activities of colony formation and tumour initiation are increased in metastatic compared to early samples, and predict metastasis in vivo. These results suggest that breast stem cell activity will predict for poor outcome tumours, and therapy targeting this activity will improve outcomes for patients with metastatic disease.

Screening for intermediate and severe forms of thalassaemia in discarded red blood cells: optimization and feasibility.

PubMed

George, Elizabeth; Lai, Mei I; Teh, Lai Kuan; Ramasamy, Rajesh; Goh, Ern Huei; Asokan, Kamalan; Tan, J A M A; Vasudevan, Maithili; Low, Sharon

2011-12-01

Detection and quantification of Hb subtypes of human blood is integral to presumptive identification of thalassaemias. It has been used in neonatal screening of thalassaemia and Hb variants. The use of discarded red blood cells following processing of the cord blood for stem cells provides readily available diagnostic material for thalassaemia screening. In this study, we determined the range of Hb subtypes in 195 consecutive cord blood samples collected for cord blood banking. The 'cord blood samples' analysed were those of the remaining red blood cells after the cord blood was processed for stem cell storage. Quantification of Hb subtypes by high performance liquid chromatography (HPLC) was done on BioRad Variant II Hb testing system. Only 73 (36.5%) of the samples could be analyzed neat without dilution. With a 1:300 dilution with wash solution the acceptable area as recommended by the manufacturer for reading of a C-gram within the 1 to 3 million ranges were achieved in all. Eighteen (9%) 12 showed classical Hb Barts (y4) prerun peaks were confirmed by Sebia Hydrasys automated Hb gel electrophoresis and quantified by Sebia Capillarys 2 capillary electrophoresis. Only 1 (0.5%) was presumptively identified with HbH disease. Due to the limited number of samples no beta-thalassaemia major, Hb E beta-thalassaemia and Hb Barts hydrops fetalis were found. The HPLC assay was possible at a cost US$ 5 per sample and a turnover time of 10 samples per hour without technical difficulties. This study reports an effective and valuable protocol for thalassaemia screening in red blood cells which would otherwise be discarded during cord blood processing. Cord blood with severe and intermediate forms of thalassaemia can be preselected and not stored.

Fluorescence- and magnetic-activated cell sorting strategies to separate spermatozoa involving plural contributors from biological mixtures for human identification

PubMed Central

Xu, Yan; Xie, Jianhui; Chen, Ronghua; Cao, Yu; Ping, Yuan; Xu, Qingwen; Hu, Wei; Wu, Dan; Gu, Lihua; Zhou, Huaigu; Chen, Xin; Zhao, Ziqin; Zhong, Jiang; Li, Rui

2016-01-01

No effective method has been developed to distinguish sperm cells originating from different men in multi-suspect sexual assault cases. Here we combined MACS and FACS to isolate single donor sperm cells from forensic mixture samples including female vaginal epithelial cells and sperm cells from multiple contributors. Sperms from vaginal swab were isolated by MACS using FITC-conjugated A kinase anchor protein 3 (AKAP3) antibody; target individual sperm cells involving two or three donors were separated by FACS using FITC-labeled blood group A/B antigen antibody. This procedure was further tested in two mock multi-suspect sexual assault samples and one practical casework sample. Our results showed that complete single donor STR profiles could be successfully obtained from sperm/epithelial cell and sperm mixtures from two contributors. For unbalanced sperm/epithelial cells and sperm cells mixtures, sensitivity results revealed that target cells could be detected at as low as 1:32 and 1:8 mixed ratios, respectively. Although highly relies on cell number and blood types or secretor status of the individuals, this procedure would still be useful tools for forensic DNA analysis of multi-suspect sexual assault cases by the combined use of FACS and MACS based on sperm-specific AKAP3 antigen and human blood type antigen. PMID:27857155

Improving public health surveillance using a dual-frame survey of landline and cell phone numbers.

PubMed

Hu, S Sean; Balluz, Lina; Battaglia, Michael P; Frankel, Martin R

2011-03-15

To meet challenges arising from increasing rates of noncoverage in US landline-based telephone samples due to cell-phone-only households, the Behavioral Risk Factor Surveillance System (BRFSS) expanded a traditional landline-based random digit dialing survey to a dual-frame survey of landline and cell phone numbers. In 2008, a survey of adults with cell phones only was conducted in parallel with an ongoing landline-based health survey in 18 states. The authors used the optimal approach to allocate samples into landline and cell-phone-only strata and used a new approach to weighting state-level landline and cell phone samples. They developed logistic models for each of 16 health indicators to examine whether exclusion of adults with cell phones only affected estimates after adjustment for demographic characteristics. The extents of the potential biases in landline telephone surveys that exclude cell phones were estimated. Biases resulting from exclusion of adults with cell phones only from the landline-based survey were found for 9 out of the 16 health indicators. Because landline noncoverage rates for adults with cell phones only continue to increase, these biases are likely to increase. Use of a dual-frame survey of landline and cell phone numbers assisted the BRFSS efforts in obtaining valid, reliable, and representative data. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health 2011.

Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry.

PubMed

Servais; Courties; Lebaron; Troussellier

1999-08-01

> Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 µm(3)). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p180.html

High-throughput rare cell separation from blood samples using steric hindrance and inertial microfluidics.

PubMed

Shen, Shaofei; Ma, Chao; Zhao, Lei; Wang, Yaolei; Wang, Jian-Chun; Xu, Juan; Li, Tianbao; Pang, Long; Wang, Jinyi

2014-07-21

The presence and quantity of rare cells in the bloodstream of cancer patients provide a potentially accessible source for the early detection of invasive cancer and for monitoring the treatment of advanced diseases. The separation of rare cells from peripheral blood, as a "virtual and real-time liquid biopsy", is expected to replace conventional tissue biopsies of metastatic tumors for therapy guidance. However, technical obstacles, similar to looking for a needle in a haystack, have hindered the broad clinical utility of this method. In this study, we developed a multistage microfluidic device for continuous label-free separation and enrichment of rare cells from blood samples based on cell size and deformability. We successfully separated tumor cells (MCF-7 and HeLa cells) and leukemic (K562) cells spiked in diluted whole blood using a unique complementary combination of inertial microfluidics and steric hindrance in a microfluidic system. The processing parameters of the inertial focusing and steric hindrance regions were optimized to achieve high-throughput and high-efficiency separation, significant advantages compared with existing rare cell isolation technologies. The results from experiments with rare cells spiked in 1% hematocrit blood indicated >90% cell recovery at a throughput of 2.24 × 10(7) cells min(-1). The enrichment of rare cells was >2.02 × 10(5)-fold. Thus, this microfluidic system driven by purely hydrodynamic forces has practical potential to be applied either alone or as a sample preparation platform for fundamental studies and clinical applications.

Fluorescent-Magnetic-Biotargeting Multifunctional Nanobioprobes for Detecting and Isolating Multiple Types of Tumor Cells

PubMed Central

Song, Er-Qun; Hu, Jun; Wen, Cong-Ying; Tian, Zhi-Quan; Yu, Xu; Zhang, Zhi-Ling; Shi, Yun-Bo; Pang, Dai-Wen

2011-01-01

Fluorescent-magnetic-biotargeting multifunctional nanobioprobes (FMBMNs) have attracted great attention in recent years due to their increasing, important applications in biomedical research, clinical diagnosis, and biomedicine. We have previously developed such nanobioprobes for the detection and isolation of a single kind of tumor cells. Detection and isolation of multiple tumor markers or tumor cells from complex samples sensitively and with high efficiency is critical for the early diagnosis of tumors, especially malignant tumors or cancers, which will improve clinical diagnosis outcomes and help to select effective treatment approaches. Here, we expanded the application of the monoclonal antibody (mAb)-coupled FMBMNs for multiplexed assays. Multiple types of cancer cells, such as leukemia cells and prostate cancer cells, were detected and collected from mixed samples within 25 minutes by using a magnet and an ordinary fluorescence microscope. The capture efficiencies of mAb-coupled FMBMNs for the above mentioned two types of cells were 96% and 97% respectively. Furthermore, by using the mAb-coupled FMBMNs, specific and sensitive detection and rapid separation of a small number of spiked leukemia cells and prostate cancer cells in a large population of cultured normal cells (about 0.01% were tumor cells) were achieved simply and inexpensively without any sample pretreatment before cell analysis. Therefore, mAb-coupled multicolour FMBMNs may be used for very sensitive detection and rapid isolation of multiple cancer cells in biomedical research and medical diagnostics. PMID:21250650

Epstein-Barr Virus Type 2 Infects T Cells in Healthy Kenyan Children.

PubMed

Coleman, Carrie B; Daud, Ibrahim I; Ogolla, Sidney O; Ritchie, Julie A; Smith, Nicholas A; Sumba, Peter O; Dent, Arlene E; Rochford, Rosemary

2017-09-15

The 2 strains of Epstein-Barr virus (EBV), EBV type 1 (EBV-1) and EBV-2, differ in latency genes, suggesting that they use distinct mechanisms to establish latency. We previously reported that EBV-2 infects T cells in vitro. In this study, we tested the possibility that EBV-2 infects T cells in vivo. Purified T-cell fractions isolated from children positive for EBV-1 or EBV-2 and their mothers were examined for the presence of EBV and for EBV type. We detected EBV-2 in all T-cell samples obtained from EBV-2-infected children at 12 months of age, with some children retaining EBV-2-positive T cells through 24 months of age, suggesting that EBV-2 persists in T cells. We were unable to detect EBV-2 in T-cell samples from mothers but could detect EBV-2 in samples of their breast milk and saliva. These data suggest that EBV-2 uses T cells as an additional latency reservoir but that, over time, the frequency of infected T cells may drop below detectable levels. Alternatively, EBV-2 may establish a prolonged transient infection in the T-cell compartment. Collectively, these novel findings demonstrate that EBV-2 infects T cells in vivo and suggest EBV-2 may use the T-cell compartment to establish latency. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer.

PubMed

Schneck, Helen; Gierke, Berthold; Uppenkamp, Frauke; Behrens, Bianca; Niederacher, Dieter; Stoecklein, Nikolas H; Templin, Markus F; Pawlak, Michael; Fehm, Tanja; Neubauer, Hans

2015-01-01

Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.

Infrared trace element detection system

DOEpatents

Bien, F.; Bernstein, L.S.; Matthew, M.W.

1988-11-15

An infrared trace element detection system includes an optical cell into which the sample fluid to be examined is introduced and removed. Also introduced into the optical cell is a sample beam of infrared radiation in a first wavelength band which is significantly absorbed by the trace element and a second wavelength band which is not significantly absorbed by the trace element for passage through the optical cell through the sample fluid. The output intensities of the sample beam of radiation are selectively detected in the first and second wavelength bands. The intensities of a reference beam of the radiation are similarly detected in the first and second wavelength bands. The sensed output intensity of the sample beam in one of the first and second wavelength bands is normalized with respect to the other and similarly, the intensity of the reference beam of radiation in one of the first and second wavelength bands is normalized with respect to the other. The normalized sample beam intensity and normalized reference beam intensity are then compared to provide a signal from which the amount of trace element in the sample fluid can be determined. 11 figs.

Infrared trace element detection system

DOEpatents

Bien, Fritz; Bernstein, Lawrence S.; Matthew, Michael W.

1988-01-01

An infrared trace element detection system including an optical cell into which the sample fluid to be examined is introduced and removed. Also introduced into the optical cell is a sample beam of infrared radiation in a first wavelength band which is significantly absorbed by the trace element and a second wavelength band which is not significantly absorbed by the trace element for passage through the optical cell through the sample fluid. The output intensities of the sample beam of radiation are selectively detected in the first and second wavelength bands. The intensities of a reference beam of the radiation are similarly detected in the first and second wavelength bands. The sensed output intensity of the sample beam in one of the first and second wavelength bands is normalized with respect to the other and similarly, the intensity of the reference beam of radiation in one of the first and second wavelength bands is normalized with respect to the other. The normalized sample beam intensity and normalized reference beam intensity are then compared to provide a signal from which the amount of trace element in the sample fluid can be determined.

Cryogenic coherent X-ray diffraction imaging of biological samples at SACLA: a correlative approach with cryo-electron and light microscopy.

PubMed

Takayama, Yuki; Yonekura, Koji

2016-03-01

Coherent X-ray diffraction imaging at cryogenic temperature (cryo-CXDI) allows the analysis of internal structures of unstained, non-crystalline, whole biological samples in micrometre to sub-micrometre dimensions. Targets include cells and cell organelles. This approach involves preparing frozen-hydrated samples under controlled humidity, transferring the samples to a cryo-stage inside a vacuum chamber of a diffractometer, and then exposing the samples to coherent X-rays. Since 2012, cryo-coherent diffraction imaging (CDI) experiments have been carried out with the X-ray free-electron laser (XFEL) at the SPring-8 Ångstrom Compact free-electron LAser (SACLA) facility in Japan. Complementary use of cryo-electron microscopy and/or light microscopy is highly beneficial for both pre-checking samples and studying the integrity or nature of the sample. This article reports the authors' experience in cryo-XFEL-CDI of biological cells and organelles at SACLA, and describes an attempt towards reliable and higher-resolution reconstructions, including signal enhancement with strong scatterers and Patterson-search phasing.

Peripheral biomarkers revisited: integrative profiling of peripheral samples for psychiatric research.

PubMed

Hayashi-Takagi, Akiko; Vawter, Marquis P; Iwamoto, Kazuya

2014-06-15

Peripheral samples, such as blood and skin, have been used for decades in psychiatric research as surrogates for central nervous system samples. Although the validity of the data obtained from peripheral samples has been questioned and other state-of-the-art techniques, such as human brain imaging, genomics, and induced pluripotent stem cells, seem to reduce the value of peripheral cells, accumulating evidence has suggested that revisiting peripheral samples is worthwhile. Here, we re-evaluate the utility of peripheral samples and argue that establishing an understanding of the common signaling and biological processes in the brain and peripheral samples is required for the validity of such models. First, we present an overview of the available types of peripheral cells and describe their advantages and disadvantages. We then briefly summarize the main achievements of omics studies, including epigenome, transcriptome, proteome, and metabolome analyses, as well as the main findings of functional cellular assays, the results of which imply that alterations in neurotransmission, metabolism, the cell cycle, and the immune system may be partially responsible for the pathophysiology of major psychiatric disorders such as schizophrenia. Finally, we discuss the future utility of peripheral samples for the development of biomarkers and tailor-made therapies, such as multimodal assays that are used as a battery of disease and trait pathways and that might be potent and complimentary tools for use in psychiatric research. © 2013 Society of Biological Psychiatry Published by Society of Biological Psychiatry All rights reserved.

Automated cell disruption is a reliable and effective method of isolating RNA from fresh snap-frozen normal and malignant oral mucosa samples.

PubMed

Van der Vorst, Sébastien; Dekairelle, Anne-France; Irenge, Léonid; Hamoir, Marc; Robert, Annie; Gala, Jean-Luc

2009-01-01

This study compared automated vs. manual tissue grinding in terms of RNA yield obtained from oral mucosa biopsies. A total of 20 patients undergoing uvulectomy for sleep-related disorders and 10 patients undergoing biopsy for head and neck squamous cell carcinoma were enrolled in the study. Samples were collected, snap-frozen in liquid nitrogen, and divided into two parts of similar weight. Sample grinding was performed on one sample from each pair, either manually or using an automated cell disruptor. The performance and efficacy of each homogenization approach was compared in terms of total RNA yield (spectrophotometry, fluorometry), mRNA quantity [densitometry of specific TP53 amplicons and TP53 quantitative reverse-transcribed real-time PCR (qRT-PCR)], and mRNA quality (functional analysis of separated alleles in yeast). Although spectrophotometry and fluorometry results were comparable for both homogenization methods, TP53 expression values obtained by amplicon densitometry and qRT-PCR were significantly and consistently better after automated homogenization (p<0.005) for both uvula and tumor samples. Functional analysis of separated alleles in yeast results was better with the automated technique for tumor samples. Automated tissue homogenization appears to be a versatile, quick, and reliable method of cell disruption and is especially useful in the case of small malignant samples, which show unreliable results when processed by manual homogenization.

Experimental set up for the irradiation of biological samples and nuclear track detectors with UV C

PubMed Central

Portu, Agustina Mariana; Rossini, Andrés Eugenio; Gadan, Mario Alberto; Bernaola, Omar Alberto; Thorp, Silvia Inés; Curotto, Paula; Pozzi, Emiliano César Cayetano; Cabrini, Rómulo Luis; Martin, Gisela Saint

2016-01-01

Aim In this work we present a methodology to produce an “imprint” of cells cultivated on a polycarbonate detector by exposure of the detector to UV C radiation. Background The distribution and concentration of 10B atoms in tissue samples coming from BNCT (Boron Neutron Capture Therapy) protocols can be determined through the quantification and analysis of the tracks forming its autoradiography image on a nuclear track detector. The location of boron atoms in the cell structure could be known more accurately by the simultaneous observation of the nuclear tracks and the sample image on the detector. Materials and Methods A UV C irradiator was constructed. The irradiance was measured along the lamp direction and at different distances. Melanoma cells were cultured on polycarbonate foils, incubated with borophenylalanine, irradiated with thermal neutrons and exposed to UV C radiation. The samples were chemically attacked with a KOH solution. Results A uniform irradiation field was established to expose the detector foils to UV C light. Cells could be seeded on the polycarbonate surface. Both imprints from cells and nuclear tracks were obtained after chemical etching. Conclusions It is possible to yield cellular imprints in polycarbonate. The nuclear tracks were mostly present inside the cells, indicating a preferential boron uptake. PMID:26933396

Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples

PubMed Central

Kopek, Benjamin G.; Paez-Segala, Maria G.; Shtengel, Gleb; Sochacki, Kem A.; Sun, Mei G.; Wang, Yalin; Xu, C. Shan; van Engelenburg, Schuyler B.; Taraska, Justin W.; Looger, Loren L.; Hess, Harald F.

2017-01-01

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM datasets on aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. Choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replica creates high-contrast, 3-dimensional images of the cytoplasmic surface of the plasma membrane, but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples, but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (~10–50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2–7 days, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology. PMID:28384138

Effect of CNT as a Nucleating Agent on Cell Morphology and Thermal Insulation Property of the Rigid Polyurethane Foams.

PubMed

Ahn, WonSool; Lee, Joon-Man

2015-11-01

The effects of MWCNT on the cell sizes, cell uniformities, thermal conductivities, bulk densities, foaming kinetics, and compressive mechanical properties of the rigid PUFs were investigated. To obtain the better uniform dispersed state of MWCNT, grease-type master batch of MWCNT/surfactant was prepared by three-roll mill. Average cell size of the PUF samples decreased from 185.1 for the neat PUF to 162.9 μm for the sample of 0.01 phr of MWCNT concentration. Cell uniformity was also enhanced showing the standard cell-size deviation of 61.7 and 35.2, respectively. While the thermal conductivity of the neat PUF was 0.0222 W/m(o)K, that of the sample with 0.01 phr of MWCNT showed 0.0204 W/m(o)K, resulting 8.2% reduction of the thermal conductivity. Bulk density of the PUF samples was observed as nearly the same values as 30.0 ± 1.0 g/cm3 regardless of MWCNT. Temperature profiles during foaming process showed that an indirect indication of the nucleation effect of MWCNT for the PUF foaming system, showing faster and higher temperature rising with time. The compressive yield stress is nearly the same as 0.030 x 10(5) Pa regardless of MWCNT.

The role of electron irradiation history in liquid cell transmission electron microscopy.

PubMed

Moser, Trevor H; Mehta, Hardeep; Park, Chiwoo; Kelly, Ryan T; Shokuhfar, Tolou; Evans, James E

2018-04-01

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.

The role of electron irradiation history in liquid cell transmission electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC- TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the rolemore » of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

The role of electron irradiation history in liquid cell transmission electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role ofmore » cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. Lastly, these results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

Enhanced human osteoblast cell adhesion and proliferation on 316 LS stainless steel by means of CO2 laser surface treatment.

PubMed

Hao, L; Lawrence, J; Phua, Y F; Chian, K S; Lim, G C; Zheng, H Y

2005-04-01

An effective and novel technique for improving the biocompatibility of a biograde 316 LS stainless steel through the application of CO(2) laser treatment to modify the surface properties of the material is described herein. Different surface properties, such as surface roughness, surface oxygen content, and surface energy for CO(2) laser-treated 316 LS stainless steel, untreated, and mechanically roughened samples were analyzed, and their effects on the wettability characteristics of the material were studied. It was found that modification of the wettability characteristics of the 316 LS stainless steel following CO(2) laser treatment was achieved. This improvement was identified as being mainly due to the change in the polar component of the surface energy. One-day cell adhesion tests showed that cells not only adhered and spread better, but also grew faster on the CO(2) laser-treated sample than on either the untreated or mechanically roughened sample. Further, compared with the untreated sample, MTT cell proliferation analysis revealed that the mechanically roughed surface resulted in a slight enhancement, and CO(2) laser treatment brought about a significant increase in cell proliferation. An increase in the wettability of the 316 LS stainless steel was observed to positively correlate with the cell proliferation. (c) 2004 Wiley Periodicals, Inc.

Sprouty2 protein is downregulated in human squamous cell carcinoma of the head and neck and suppresses cell proliferation in vitro.

PubMed

Lin, Chiang-Liang; Chiang, Wei-Fan; Tung, Chao-Ling; Hsieh, Jeng-Long; Hsiao, Jenn-Ren; Huang, Wen-Tsung; Feng, Li-Yia; Chang, Chi-Hua; Liu, Shyun-Yeu; Tsao, Chao-Jung; Feng, Yin-Hsun

2015-01-01

Sprouty2 is known for its tumor-suppressing effect in various human malignant diseases. In head and neck squamous cell carcinoma (HNSCC), the role of sprouty2 in tumorigenesis and clinical implication remains elusive. The aim of the present study was to investigate the expression of sprouty2 in patients with HNSCC and its function in vitro. Quantitative analysis of mRNA expression of sprouty2 was performed on frozen tumor samples from 42 patients with HNSCC and 19 with oral verrucous hyperplasia (OVH) with paired counterparts of normal mucosa. Downregulation of sprouty2 expression was demonstrated in 79% of HNSCC samples and in 58% of OVH samples compared with paired samples of normal mucosa. Enhanced expression of sprouty2 protein suppressed the growth of HNSCC cells and signaling of the phosphorylated AKT pathway. Following transfection of the sprouty2 plasmid, HNSCC cells were more sensitive to sorafenib, a tyrosine kinase inhibitor of Raf and vascular endothelial growth factor receptor. The present study suggested that sprouty2 expression was downregulated and behaved as a tumor suppressor in HNSCC. Sprouty2 expression in tumor cells enhanced sensitivity to sorafenib. Further studies are required to define the clinical impact of sprouty2 in patients with HNSCC.

The role of electron irradiation history in liquid cell transmission electron microscopy

PubMed Central

Mehta, Hardeep

2018-01-01

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides. PMID:29725619

The role of electron irradiation history in liquid cell transmission electron microscopy

DOE PAGES

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo; ...

2018-04-20

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role ofmore » cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. Lastly, these results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

Intracellular flow cytometry may be combined with good quality and high sensitivity RT-qPCR analysis.

PubMed

Sandstedt, Mikael; Jonsson, Marianne; Asp, Julia; Dellgren, Göran; Lindahl, Anders; Jeppsson, Anders; Sandstedt, Joakim

2015-12-01

Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples. © 2015 International Society for Advancement of Cytometry.

Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana

PubMed Central

Schliep, Martin; Ebert, Berit; Simon-Rosin, Ulrike; Zoeller, Daniela

2010-01-01

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes. PMID:20101514

Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana.

PubMed

Schliep, Martin; Ebert, Berit; Simon-Rosin, Ulrike; Zoeller, Daniela; Fisahn, Joachim

2010-05-01

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes.

A Paper-Based Electrochromic Array for Visualized Electrochemical Sensing.

PubMed

Zhang, Fengling; Cai, Tianyi; Ma, Liang; Zhan, Liyuan; Liu, Hong

2017-01-31

We report a battery-powered, paper-based electrochromic array for visualized electrochemical sensing. The paper-based sensing system consists of six parallel electrochemical cells, which are powered by an aluminum-air battery. Each single electrochemical cell uses a Prussian Blue spot electrodeposited on an indium-doped tin oxide thin film as the electrochromic indicator. Each electrochemical cell is preloaded with increasing amounts of analyte. The sample activates the battery for the sensing. Both the preloaded analyte and the analyte in the sample initiate the color change of Prussian Blue to Prussian White. With a reaction time of 60 s, the number of electrochemical cells with complete color changes is correlated to the concentration of analyte in the sample. As a proof-of-concept analyte, lactic acid was detected semi-quantitatively using the naked eye.

Devices and process for high-pressure magic angle spinning nuclear magnetic resonance

DOEpatents

Hoyt, David W; Sears, Jr., Jesse A; Turcu, Romulus V.F.; Rosso, Kevin M; Hu, Jian Zhi

2014-04-08

A high-pressure magic angle spinning (MAS) rotor is detailed that includes a high-pressure sample cell that maintains high pressures exceeding 150 bar. The sample cell design minimizes pressure losses due to penetration over an extended period of time.

Devices and process for high-pressure magic angle spinning nuclear magnetic resonance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoyt, David W.; Sears, Jesse A.; Turcu, Romulus V. F.

A high-pressure magic angle spinning (MAS) rotor is detailed that includes a high-pressure sample cell that maintains high pressures exceeding 150 bar. The sample cell design minimizes pressure losses due to penetration over an extended period of time.

Mast cells and matrix metalloproteinase 9 expression in actinic cheilitis and lip squamous cell carcinoma.

PubMed

Souza Freitas, Valéria; de Andrade Santos, Pedro Paulo; de Almeida Freitas, Roseana; Pereira Pinto, Leão; de Souza, Lélia Batista

2011-09-01

The aim of this study was to evaluate mast cell (MC) density and migration and their association with matrix metalloproteinase (MMP) 9 expression in squamous cell carcinoma (SCC) and actinic cheilitis (AC). Tryptase, c-Kit, and MMP-9 expression was evaluated in 20 cases of SCC, 20 cases of AC, and 7 cases of normal lip (control samples) by immunohistochemistry techniques. Tryptase(+) and c-Kit(+) MC densities were significantly higher in SCCs than in ACs and control samples (P < .001). However, no significant difference was found when comparing tryptase(+) and c-Kit(+) MC densities between ACs and control samples (P values .185 and .516, respectively). MMP-9 was strongly expressed in SCCs and moderately expressed in ACs and control samples. A highly significant association was found between tryptase(+) MC density and the expression of MMP-9 (P < .001). The increase in MC density associated with the strong expression of MMP-9 may favor SCC progression. Copyright © 2011 Mosby, Inc. All rights reserved.

AuNP-DG: deoxyglucose-labeled gold nanoparticles as X-ray computed tomography contrast agents for cancer imaging.

PubMed

Aydogan, Bulent; Li, Ji; Rajh, Tijana; Chaudhary, Ahmed; Chmura, Steven J; Pelizzari, Charles; Wietholt, Christian; Kurtoglu, Metin; Redmond, Peter

2010-10-01

To study the feasibility of using 2-deoxy-D-glucose (2-DG)-labeled gold nanoparticle (AuNP-DG) as a computed tomography (CT) contrast agent with tumor targeting capability through in vitro experiments. Gold nanoparticles (AuNP) were fabricated and were conjugated with 2-deoxy-D-glucose. The human alveolar epithelial cancer cell line, A-549, was chosen for the in vitro cellular uptake assay. Two groups of cell samples were incubated with the AuNP-DG and the unlabeled AuNP, respectively. Following the incubation, the cells were washed with sterile PBS to remove the excess gold nanoparticles and spun to cell pellets using a centrifuge. The cell pellets were imaged using a microCT scanner immediately after the centrifugation. The reconstructed CT images were analyzed using a commercial software package. Significant contrast enhancement in the cell samples incubated with the AuNP-DG with respect to the cell samples incubated with the unlabeled AuNP was observed in multiple CT slices. Results from this study demonstrate enhanced uptake of 2-DG-labeled gold nanoparticle by cancer cells in vitro and warrant further experiments to study the exact molecular mechanism by which the AuNP-DG is internalized and retained in the tumor cells.

Expression of insulin-like growth factor-1 and proliferating cell nuclear antigen in human pulp cells of teeth with complete and incomplete root development.

PubMed

Caviedes-Bucheli, J; Canales-Sánchez, P; Castrillón-Sarria, N; Jovel-Garcia, J; Alvarez-Vásquez, J; Rivero, C; Azuero-Holguín, M M; Diaz, E; Munoz, H R

2009-08-01

To quantify the expression of insulin-like growth factor-1 (IGF-1) and proliferating cell nuclear antigen (PCNA) in human pulp cells of teeth with complete or incomplete root development, to support the specific role of IGF-1 in cell proliferation during tooth development and pulp reparative processes. Twenty six pulp samples were obtained from freshly extracted human third molars, equally divided in two groups according to root development stage (complete or incomplete root development). All samples were processed and immunostained to determine the expression of IGF-1 and PCNA in pulp cells. Sections were observed with a light microscope at 80x and morphometric analyses were performed to calculate the area of PCNA and IGF-1 immunostaining using digital image software. Mann-Whitney's test was used to determine statistically significant differences between groups (P < 0.05) for each peptide and the co-expression of both. Expression of IGF-1 and PCNA was observed in all human pulp samples with a statistically significant higher expression in cells of pulps having complete root development (P = 0.0009). Insulin-like growth factor-1 and PCNA are expressed in human pulp cells, with a significant greater expression in pulp cells of teeth having complete root development.

Damage coefficients in low resistivity silicon. [solar cells

NASA Technical Reports Server (NTRS)

Srour, J. R.; Othmer, S.; Chiu, K. Y.; Curtis, O. L., Jr.

1975-01-01

Electron and proton damage coefficients are determined for low resistivity silicon based on minority-carrier lifetime measurements on bulk material and diffusion length measurements on solar cells. Irradiations were performed on bulk samples and cells fabricated from four types of boron-doped 0.1 ohm-cm silicon ingots, including the four possible combinations of high and low oxygen content and high and low dislocation density. Measurements were also made on higher resistivity boron-doped bulk samples and solar cells. Major observations and conclusions from the investigation are discussed.

Testing of the cytotoxic effects of sulfate pulp mill waste waters.

PubMed

Cernáková, M; Golis, E

1994-01-01

The effect of 22 technological waste water samples and of some standards was tested on bacteria, fungi, chlorococcal algae, flagellata, plant cells, cells of Tubifex tubifex, hamster cells V79 and the fish Lebistes reticulatus. Of these 22 samples, some inhibition of cell life processes was displayed by the black liquor formed in the production of paper pulp and viscose pulp, by the waste solution produced during the preparation of bleaching agents for paper pulp and viscose pulp, and by the residual liquor after hypochlorite treatment of paper pulp.

[Morphological changes in tongue cancer after cryosurgery].

PubMed

Zhou, X D; Mao, T Q

1993-01-01

Tca 8113 (human tongue cancer cell line) cell transplanted tumors in nude mice were treated with cryosurgery for three freeze-thaw cycles. Tumor samples were obtained by biopsies pre- and post-cryosurgery for morphological study. The results showed intercellular adhesion damage, nuclear pyknosis, cell death, etc. One week after, the deep parts of the frozen samples were similar to that of the untreated ones. Our study indicates the change of biomembrance may be also important as of nuclei in cell death and may play an important role in the treatment of cancer by cryochemistry.

Effect of cryopreservation of peripheral blood mononuclear cells (PBMCs) on the variability of an antigen-specific memory B cell ELISpot.

PubMed

Trück, Johannes; Mitchell, Ruth; Thompson, Amber J; Morales-Aza, Begonia; Clutterbuck, Elizabeth A; Kelly, Dominic F; Finn, Adam; Pollard, Andrew J

2014-01-01

The ELISpot assay is used in vaccine studies for the quantification of antigen-specific memory B cells (B(MEM)), and can be performed using cryopreserved samples. The effects of cryopreservation on B(MEM) detection and the consistency of cultured ELISpot assays when performed by different operators or laboratories are unknown. In this study, blood was taken from healthy volunteers, and a cultured ELISpot assay was used to count B(MEM) specific for 2 routine vaccine antigens (diphtheria and tetanus toxoid). Results were assessed for intra- and inter-operator variation, and the effects of cryopreservation. Cryopreserved samples were shipped to a second laboratory in order to assess inter-laboratory variation. B(MEM) frequencies were very strongly correlated when comparing fresh and frozen samples processed by the same operator, and were also very strongly correlated when comparing 2 operators in the same laboratory. Results were slightly less consistent when samples were processed in different laboratories but correlation between the 2 measurements was still very strong. Although cell viability was reduced in some cryopreserved samples due to higher temperatures during transportation, B(MEM) could still be quantified. These results demonstrate the reproducibility of the ELISpot assay across operators and laboratories, and support the use of cryopreserved samples in future B(MEM) studies.

Focusing polycapillary to reduce parasitic scattering for inelastic x-ray measurements at high pressure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chow, P., E-mail: pchow@carnegiescience.edu; Xiao, Y. M.; Rod, E.

2015-07-15

The double-differential scattering cross-section for the inelastic scattering of x-ray photons from electrons is typically orders of magnitude smaller than that of elastic scattering. With samples 10-100 μm size in a diamond anvil cell at high pressure, the inelastic x-ray scattering signals from samples are obscured by scattering from the cell gasket and diamonds. One major experimental challenge is to measure a clean inelastic signal from the sample in a diamond anvil cell. Among the many strategies for doing this, we have used a focusing polycapillary as a post-sample optic, which allows essentially only scattered photons within its input fieldmore » of view to be refocused and transmitted to the backscattering energy analyzer of the spectrometer. We describe the modified inelastic x-ray spectrometer and its alignment. With a focused incident beam which matches the sample size and the field of view of polycapillary, at relatively large scattering angles, the polycapillary effectively reduces parasitic scattering from the diamond anvil cell gasket and diamonds. Raw data collected from the helium exciton measured by x-ray inelastic scattering at high pressure using the polycapillary method are compared with those using conventional post-sample slit collimation.« less